Thrombospondin-related Adhesive Protein Research Articles

Naturally acquired immune responses to thrombospondin-related adhesion protein (TRAP) of Plasmodium vivax in patients from areas of unstable malaria transmission

A key tool for the control, elimination, and eradication of Plasmodium vivax is the development of an effective vaccine. The thrombospondin-related adhesion protein (TRAP) is one of the major sporozoite antigens that plays an important role in the invasion of mosquito salivary glands and hepatocytes by sporozoites. The main goal of this study was to evaluate the naturally acquired antibodies to the P. vivax TRAP (PvTRAP) in patients from malaria-endemic areas of Iran (n=116), Afghanistan (n=50), and Pakistan (n=50). The PvTRAP gene was expressed in Escherichia coli Rosetta (DE3)-pET23a and used as antigen in enzyme-linked immunosorbent assay (ELISA). The profile of immunoglobulin G (IgG) isotype and the avidity of IgG, IgG1, and IgG3 to PvTRAP, as well as the association between anti-PvTRAP isotype responses and host age were evaluated. Only 42.24% of Iranian, 38% of Afghani, and 44% of Pakistani patients infected with P. vivax had positive anti-PvTRAP IgG, and the prevalence of responders in the three countries did not differ significantly (P>0.05). Moreover, the prevalence of IgG1 and IgG3 antibody responses to PvTRAP showed no significant correlation with age (P>0.05). Individuals exposed to vivax malaria in the unstable malaria transmission areas are able to produce antibodies to the TRAP antigen at all ages in response to P. vivax infections. Finally, the presence of mature IgG1 and IgG3 antibodies with high to intermediate avidity against PvTRAP antigen (>60%) provide more information to understand the interactions between the host and P. vivax parasite. In summary, the present study provides data that support the rational development of an effective pre-erythrocytic stage vaccine based on PvTRAP antigen.

Virus-Like Particle (VLP) Plus Microcrystalline Tyrosine (MCT) Adjuvants Enhance Vaccine Efficacy Improving T and B Cell Immunogenicity and Protection against Plasmodium berghei/vivax

Vaccination is the most effective prophylactic tool against infectious diseases. Despite continued efforts to control malaria, the disease still generally represents a significant unmet medical need. Microcrystalline tyrosine (MCT) is a well described depot used in licensed allergy immunotherapy products and in clinical development. However, its proof of concept in prophylactic vaccines has only recently been explored. MCT has never been used in combination with virus-like particles (VLPs), which are considered to be one of the most potent inducers of cellular and humoral immune responses in mice and humans. In the current study we assessed the potential of MCT to serve as an adjuvant in the development of a vaccine against malaria either alone or combined with VLP using Plasmodium vivax thrombospondin-related adhesive protein (TRAP) as a target antigen. We chemically coupled PvTRAP to VLPs derived from the cucumber mosaic virus fused to a universal T-cell epitope of the tetanus toxin (CMVtt), formulated with MCT and compared the induced immune responses to PvTRAP formulated in PBS or Alum. The protective capacity of the various formulations was assessed using Plasmodium berghei expressing PvTRAP. All vaccine formulations using adjuvants and/or VLP increased humoral immunogenicity for PvTRAP compared to the antigen alone. The most proficient responder was the group of mice immunized with the vaccine formulated with PvTRAP-VLP + MCT. The VLP-based vaccine formulated in MCT also induced the strongest T cell response and conferred best protection against challenge with recombinant Plasmodium berghei. Thus, the combination of VLP with MCT may take advantage of the properties of each component and appears to be an alternative biodegradable depot adjuvant for development of novel prophylactic vaccines.

Viral Vector Malaria Vaccines Induce High-Level T Cell and Antibody Responses in West African Children and Infants

Heterologous prime-boosting with viral vectors encoding the pre-erythrocytic antigen thrombospondin-related adhesion protein fused to a multiple epitope string (ME-TRAP) induces CD8+ T cell-mediated immunity to malaria sporozoite challenge in European malaria-naive and Kenyan semi-immune adults. This approach has yet to be evaluated in children and infants. We assessed this vaccine strategy among 138 Gambian and Burkinabe children in four cohorts: 2- to 6-year olds in The Gambia, 5- to 17-month-olds in Burkina Faso, and 5- to 12-month-olds and 10-week-olds in The Gambia. We assessed induction of cellular immunity, taking into account the distinctive hematological status of young infants, and characterized the antibody response to vaccination. T cell responses peaked 7 days after boosting with modified vaccinia virus Ankara (MVA), with highest responses in infants aged 10 weeks at priming. Incorporating lymphocyte count into the calculation of T cell responses facilitated a more physiologically relevant comparison of cellular immunity across different age groups. Both CD8+ and CD4+ T cells secreted cytokines. Induced antibodies were up to 20-fold higher in all groups compared with Gambian and United Kingdom (UK) adults, with comparable or higher avidity. This immunization regimen elicited strong immune responses, particularly in young infants, supporting future evaluation of efficacy in this key target age group for a malaria vaccine.

Interferon-γ responses to Plasmodium falciparum vaccine candidate antigens decrease in the absence of malaria transmission.

BackgroundMalaria elimination campaigns are planned or active in many countries. The effects of malaria elimination on immune responses such as antigen-specific IFN- γ responses are not well characterized.MethodsIFN- γ responses to the P. falciparum antigens circumsporozoite protein, liver stage antigen-1, thrombospondin-related adhesive protein, apical membrane antigen-1, MB2, and merozoite surface protein-1 were tested by ELISA in 243 individuals in highland Kenya in April 2008, October 2008, and April 2009, after a one-year period of interrupted malaria transmission from April 2007 to March 2008.ResultsWhile one individual (0.4%) tested positive for P. falciparum by PCR inOctober 2008 and another two (0.9%) tested positive in April 2009, no clinical malaria cases were detected during weekly visits. Levels of IFN-γ to all antigens decreased significantly from April 2008 to April 2009 (all P < 0.001).DiscussionNaturally acquired IFN- γ responses to P. falciparum antigensare short-lived in the absence of repeated P. falciparum infection. Even short periods of malaria interruption may significantly decrease IFN-γ responses to P. falciparum antigens.

Safety, Immunogenicity and Efficacy of Prime-Boost Vaccination with ChAd63 and MVA Encoding ME-TRAP against Plasmodium falciparum Infection in Adults in Senegal.

Malaria transmission is in decline in some parts of Africa, partly due to the scaling up of control measures. If the goal of elimination is to be achieved, additional control measures including an effective and durable vaccine will be required. Studies utilising the prime-boost approach to deliver viral vectors encoding the pre-erythrocytic antigen ME-TRAP (multiple epitope thrombospondin-related adhesion protein) have shown promising safety, immunogenicity and efficacy in sporozoite challenge studies. More recently, a study in Kenyan adults, similar to that reported here, showed substantial efficacy against P. falciparum infection. One hundred and twenty healthy male volunteers, living in a malaria endemic area of Senegal were randomised to receive either the Chimpanzee adenovirus (ChAd63) ME-TRAP as prime vaccination, followed eight weeks later by modified vaccinia Ankara (MVA) also encoding ME-TRAP as booster, or two doses of anti-rabies vaccine as a comparator. Prior to follow-up, antimalarials were administered to clear parasitaemia and then participants were monitored by PCR for malaria infection for eight weeks. The primary endpoint was time-to-infection with P. falciparum malaria, determined by two consecutive positive PCR results. Secondary endpoints included adverse event reporting, measures of cellular and humoral immunogenicity and a meta-analysis of combined vaccine efficacy with the parallel study in Kenyan adults.We show that this pre-erythrocytic malaria vaccine is safe and induces significant immunogenicity, with a peak T-cell response at seven days after boosting of 932 Spot Forming Cells (SFC)/106 Peripheral Blood Mononuclear Cells(PBMC) compared to 57 SFC/ 106 PBMCs in the control group. However, a vaccine efficacy was not observed: 12 of 57 ME-TRAP vaccinees became PCR positive during the intensive monitoring period as compared to 13 of the 58 controls (P = 0.80). This trial confirms that vaccine efficacy against malaria infection in adults may be rapidly assessed using this efficient and cost-effective clinical trial design. Further efficacy evaluation of this vectored candidate vaccine approach in other malaria transmission settings and age-de-escalation into the main target age groups for a malaria vaccine is in progress.

Discriminating Protective from Nonprotective Plasmodium-Specific CD8+ T Cell Responses

Despite decades of research, malaria remains a global health crisis. Current subunit vaccine approaches do not provide efficient long-term, sterilizing immunity against Plasmodium infections in humans. Conversely, whole parasite vaccinations with their larger array of target Ags have conferred long-lasting sterilizing protection to humans. Similar studies in rodent models of malaria reveal that CD8(+) T cells play a critical role in liver-stage immunity after whole parasite vaccination. However, it is unknown whether all CD8(+) T cell specificities elicited by whole parasite vaccination contribute to protection, an issue of great relevance for enhanced subunit vaccination. In this article, we show that robust CD8(+) T cell responses of similar phenotype are mounted after prime-boost immunization against Plasmodium berghei glideosome-associated protein 5041-48-, sporozoite-specific protein 20318-325-, thrombospondin-related adhesion protein (TRAP) 130-138-, or circumsporozoite protein (CSP) 252-260-derived epitopes in mice, but only CSP252-260- and TRAP130-138-specific CD8(+) T cells provide sterilizing immunity and reduce liver parasite burden after sporozoite challenge. Further, CD8(+) T cells specific to sporozoite surface-expressed CSP and TRAP proteins, but not intracellular glideosome-associated protein 50 and sporozoite-specific protein 20, efficiently recognize sporozoite-infected hepatocytes in vitro. These results suggest that: 1) protection-relevant antigenic targets, regardless of their immunogenic potential, must be efficiently presented by infected hepatocytes for CD8(+) T cells to eliminate liver-stage Plasmodium infection; and 2) proteins expressed on the surface of sporozoites may be good target Ags for protective CD8(+) T cells.

Molecular detection and genetic diversity of Babesia gibsoni in dogs in India

Babesia gibsoni is a tick borne intraerythrocytic protozoan parasite causing piroplasmosis in dogs and has been predominantly reported in Asian countries, including Japan, Korea, Taiwan, Malaysia, Bangladesh and India. The present communication is the first evidence on the genetic diversity of B. gibsoni of dogs in India. Blood samples were collected from 164 dogs in north and northeast states of India and 13 dogs (7.9%) were found positive for B. gibsoni infection by microscopic examination of blood smears. Molecular confirmation of these microscopic positive cases for B. gibsoni was carried out by 18S rRNA nested-PCR, followed by sequencing. Nested-PCR for the 18S rRNA gene was also carried out on microscopically B. gibsoni negative samples that detected a higher percentage of dogs (28.6%) infected with B. gibsoni. Genetic diversity in B. gibsoni in India was determined by studying B. gibsoni thrombospondin-related adhesive protein (BgTRAP) gene fragments (855bp) in 19 isolates from four north and northeast states of India. Phylogenetic analysis of the BgTRAP gene revealed that B. gibsoni parasite in India and Bangladesh formed a distinct cluster away from other Asian B. gibsoni isolates available from Japan, Taiwan and Korea. In addition, tandem repeat analysis of the BgTRAP gene clearly showed considerable genetic variation among Indian isolates that was shared by B. gibsoni isolates of Bangladesh. These results suggested that B. gibsoni parasites in a different genetic clade are endemic in dogs in India and Bangladesh. Further studies are required for better understanding of the genetic diversity of B. gibsoni prevalent in India and in its neighbouring countries.

Tailoring a Combination Preerythrocytic Malaria Vaccine.

The leading malaria vaccine candidate, RTS,S, based on the Plasmodium falciparum circumsporozoite protein (CSP), will likely be the first publicly adopted malaria vaccine. However, this and other subunit vaccines, such as virus-vectored thrombospondin-related adhesive protein (TRAP), provide only intermediate to low levels of protection. In this study, the Plasmodium berghei homologues of antigens CSP and TRAP are combined. TRAP is delivered using adenovirus- and vaccinia virus-based vectors in a prime-boost regime. Initially, CSP is also delivered using these viral vectors; however, a reduction of anti-CSP antibodies is seen when combined with virus-vectored TRAP, and the combination is no more protective than either subunit vaccine alone. Using an adenovirus-CSP prime, protein-CSP boost regime, however, increases anti-CSP antibody titers by an order of magnitude, which is maintained when combined with virus-vectored TRAP. This combination regime using protein CSP provided 100% protection in C57BL/6 mice compared to no protection using virus-vectored TRAP alone and 40% protection using adenovirus-CSP prime and protein-CSP boost alone. This suggests that a combination of CSP and TRAP subunit vaccines could enhance protection against malaria.

Cryptosporidium proliferans n. sp. (Apicomplexa: Cryptosporidiidae): Molecular and Biological Evidence of Cryptic Species within Gastric Cryptosporidium of Mammals.

The morphological, biological, and molecular characteristics of Cryptosporidium muris strain TS03 are described, and the species name Cryptosporidium proliferans n. sp. is proposed. Cryptosporidium proliferans obtained from a naturally infected East African mole rat (Tachyoryctes splendens) in Kenya was propagated under laboratory conditions in rodents (SCID mice and southern multimammate mice, Mastomys coucha) and used in experiments to examine oocyst morphology and transmission. DNA from the propagated C. proliferans isolate, and C. proliferans DNA isolated from the feces of an African buffalo (Syncerus caffer) in Central African Republic, a donkey (Equus africanus) in Algeria, and a domestic horse (Equus caballus) in the Czech Republic were used for phylogenetic analyses. Oocysts of C. proliferans are morphologically distinguishable from C. parvum and C. muris HZ206, measuring 6.8–8.8 (mean = 7.7 μm) × 4.8–6.2 μm (mean = 5.3) with a length to width ratio of 1.48 (n = 100). Experimental studies using an isolate originated from T. splendens have shown that the course of C. proliferans infection in rodent hosts differs from that of C. muris and C. andersoni. The prepatent period of 18–21 days post infection (DPI) for C. proliferans in southern multimammate mice (Mastomys coucha) was similar to that of C. andersoni and longer than the 6–8 DPI prepatent period for C. muris RN66 and HZ206 in the same host. Histopatologicaly, stomach glands of southern multimammate mice infected with C. proliferans were markedly dilated and filled with necrotic material, mucus, and numerous Cryptosporidium developmental stages. Epithelial cells of infected glands were atrophic, exhibited cuboidal or squamous metaplasia, and significantly proliferated into the lumen of the stomach, forming papillary structures. The epithelial height and stomach weight were six-fold greater than in non-infected controls. Phylogenetic analyses based on small subunit rRNA, Cryptosporidium oocyst wall protein, thrombospondin-related adhesive protein of Cryptosporidium-1, heat shock protein 70, actin, heat shock protein 90 (MS2), MS1, MS3, and M16 gene sequences revealed that C. proliferans is genetically distinct from C. muris and other previously described Cryptosporidium species.

Expression of Babesia gibsoni thrombospondin-related adhesive protein in Toxoplasma gondii and evaluation of its antigenicity and immunogenicity

Expression of <i>Babesia gibsoni</i> thrombospondin-related adhesive protein in <i>Toxoplasma gondii</i> and evaluation of its antigenicity and immunogenicity

Genetic variations of four immunodominant antigens of Babesia gibsoni isolated from dogs in southwest Japan

Babesia gibsoni is a tick-borne apicomplexan parasite of dogs that often causes fever and hemolytic anemia. Previous reports have shown that the apical membrane antigen 1 (BgAMA1), the 50kDa surface antigen (BgP50), the secreted antigen 1 (BgSA1), and the thrombospondin-related adhesive protein (BgTRAP) are promising diagnostic antigens and vaccine candidates against B. gibsoni. In the present study, we investigated the genetic polymorphisms and natural selection of these four genes of B. gibsoni isolated from dogs in southwest Japan. The prediction B-cell epitopes showed high antigenic score in the insert and indel regions of BgSA1 and BgTRAP. Sequence analyses have revealed that BgAMA1 had the highest nucleotide diversity, followed by BgP50, BgSA1 and BgTRAP. Meanwhile, the Tajima's D value test suggested balancing selection for BgAMA1 and BgP50. However, BgSA1 and BgTRAP have purifying selection making them potential vaccine candidate and diagnostic antigens since they are highly conserved. Our findings provide the genetic basis for designing and testing the efficacy of diagnostic antigens as well as vaccine candidates against B. gibsoni.

A comparative study on worldwide genetic diversity and population structure analysis of Plasmodium vivax thrombospondin-related adhesive protein (PvTRAP) and its implications for the vivax vaccine design

Plasmodium vivax thrombospondin-related anonymous protein (PvTRAP) is a promising malaria vaccine candidate; however, it exhibits sequence heterogeneity. Therefore, to design a broadly protective vivax vaccine, it is essential to have adequate information on signatures of selection and geospatial genetic diversity of global PvTRAP. For this purpose, 50 Iranian pvtrap were sequenced and compared with related available global sequences in GenBank. The nucleotide sequence analysis of Iranian pvtrap in comparison with the Sal-1 sequence showed the occurrence of 15 SNPs, and all sites were dimorphic. In total, 12 amino acid substitutions were detected and 2 of which were novel, resulting in 10 haplotypes that 8 of them were not reported in any other geographic regions. In comparison with global population, haplotype and nucleotide diversities were lowest in South Korean populations while higher levels of diversities were observed in Thai and Brazilian P. vivax populations. All 12 amino acid replacements in ectodomain of Iranian PvTRAP were distributed in predicted either B- or T-cells epitope as well as intrinsically unstructured/disordered regions (IURs). The present results revealed that observing the relatively low-level diversity in PvTRAP protein might actually be selected by immune response. In summary, the present analysis in parallel to the limited available published data has shown that genetic diversity in the global pvtrap exhibits low-level diversity and geographic variation. These results are of practical significance for the strategic development and deployment of control measures in particular for development of PvTRAP-based malaria vaccine.

Seroprevalence survey of Babesia gibsoni infection and tick species in dogs in East China

A seroprevalence survey of Babesia gibsoni infection in dogs in East China was conducted using an ELISA with recombinant B. gibsoni thrombospondin-related adhesive protein (BgTRAP). A total of 1170 dogs from East China were examined and the seroprevalence was 9.23%. The proportion of samples was 81.2%, 7.86% and 10.94% from pet, working and fighting dogs, respectively. The fighting dogs showed highest seroprevalence (39.8%) compared with working dogs (26.1%) and pet dogs (3.47%). These results indicate that B. gibsoni infection of dogs has a widespread geographic distribution throughout East China. The dominant ticks collected from the dogs were identified as Rhipicephalus sanguineus (65.57%), Haemaphysalis longicornis (21.58%) and Rhipicephalus hemaphysaloides (10.7%). Besides adult, larval and nymph stages of ticks were also recorded on dogs. This is the first report of seroprevalence of canine B. gibsoni infection and tick species in dogs in China.

Prime-boost vaccination with chimpanzee adenovirus and modified vaccinia Ankara encoding TRAP provides partial protection against Plasmodium falciparum infection in Kenyan adults.

Protective immunity to the liver stage of the malaria parasite can be conferred by vaccine-induced T cells, but no subunit vaccination approach based on cellular immunity has shown efficacy in field studies. We randomly allocated 121 healthy adult male volunteers in Kilifi, Kenya, to vaccination with the recombinant viral vectors chimpanzee adenovirus 63 (ChAd63) and modified vaccinia Ankara (MVA), both encoding the malaria peptide sequence ME-TRAP (the multiple epitope string and thrombospondin-related adhesion protein), or to vaccination with rabies vaccine as a control. We gave antimalarials to clear parasitemia and conducted PCR (polymerase chain reaction) analysis on blood samples three times a week to identify infection with the malaria parasite Plasmodium falciparum. On Cox regression, vaccination reduced the risk of infection by 67% [95% confidence interval (CI), 33 to 83%; P = 0.002] during 8 weeks of monitoring. T cell responses to TRAP peptides 21 to 30 were significantly associated with protection (hazard ratio, 0.24; 95% CI, 0.08 to 0.75; P = 0.016).

Molecular detection and genetic diversity of Babesia gibsoni in dogs in Bangladesh

Babesia gibsoni is a tick-borne hemoprotozoan parasite of dogs that often causes fever and hemolytic illness. Detection of B. gibsoni has been predominantly reported in Asian countries, including Japan, Korea, Taiwan, Malaysia, Bangladesh and India. The present study shows the first molecular characterization of B. gibsoni detected from dogs in Bangladesh. Blood samples were collected on FTA® Elute cards from 50 stray dogs in Mymensingh District in Bangladesh. DNA eluted from the cards was subjected to nested PCR for the 18S rRNA gene of Babesia species. Approximately 800bp PCR products were detected in 15 of 50 dogs (30%). Based on restriction fragment length polymorphism (RFLP) and direct sequencing of the PCR products, all parasite isolates were identified as B. gibsoni. Furthermore, the BgTRAP (B. gibsoni thrombospondin-related adhesive protein) gene fragments were detected in 13 of 15 18S rRNA gene PCR positive blood samples. Phylogenetic analysis of the BgTRAP gene revealed that B. gibsoni parasites in Bangladesh formed a cluster, which was genetically different from other Asian B. gibsoni isolates. In addition, tandem repeat analysis of the BgTRAP gene clearly showed considerable genetic variation among Bangladeshi isolates. These results suggested that B. gibsoni parasites in a different genetic clade are endemic in dogs in Bangladesh. Further studies are required to elucidate the origin, distribution, vector and pathogenesis of B. gibsoni parasites circulating in dogs in Bangladesh.

Identification and Characterization of Protein-Protein Interaction Effectors Targeting the Invasion Machinery of the Malaria Parasite

Emerging resistance of Plasmodium falciparum to current treatments is concerning and requires the development of a new generation of drugs. In pursuit of novel compounds, we have targeted protein-protein interactions (PPIs) essential to the parasite. Plasmodium uses an actomyosin motor, part of the glideosome complex, for gliding and invasion of host cells. A key interaction of the glideosome is between aldolase and the cytoplasmic tail of the thrombospondin-related adhesive protein, TRAP. It is thought that the dynamic aldolase-TRAP complex must dissociate for forward motility and invasion to progress; therefore, we have developed an assay to identify compounds that promote this interaction, resulting in stalled gliding and invasion. We produced MBP-tagged and biotinylated peptides for studying the interaction between aldolase and TRAP using surface plasmon resonance. We have screened the Medicines for Malaria Ventures library and have identified several compounds with concentration-dependent effects in vitro. Five promising compounds were moved forward into parasite gliding and invasion assays. We have observed phenotypic differences in the ability of Plasmodium parasites to glide, and found a reduced gliding velocity and abnormal rates of sporozoite attachment and detachment. To determine the effect on invasion, both red blood cell and hepatocyte invasion assays have been performed. We are currently attempting to determine a co-crystal structure of the ternary aldolase-TRAP-compound complex, and to move promising compounds into an in vivo mouse model.

Spatial variation in genetic diversity and natural selection on the thrombospondin-related adhesive protein locus of Plasmodium vivax (PvTRAP).

Thrombospondin-related adhesive protein (TRAP) of malaria parasites is essential for sporozoite motility and invasions into mosquito’s salivary gland and vertebrate’s hepatocyte; thereby, it is a promising target for pre-erythrocytic vaccine. TRAP of Plasmodium vivax (PvTRAP) exhibits sequence heterogeneity among isolates, an issue relevant to vaccine development. To gain insights into variation in the complete PvTRAP sequences of parasites in Thailand, 114 vivax malaria patients were recruited in 2006–2007 from 4 major endemic provinces bordering Myanmar (Tak in the northwest, n = 30 and Prachuap Khirikhan in the southwest, n = 25), Cambodia (Chanthaburi in the east, n = 29) and Malaysia (Yala and Narathiwat in the south, n = 30). In total, 26 amino acid substitutions were detected and 9 of which were novel, resulting in 44 distinct haplotypes. Haplotype and nucleotide diversities were lowest in southern P. vivax population while higher levels of diversities were observed in other populations. Evidences of positive selection on PvTRAP were demonstrated in domains II and IV and purifying selection in domains I, II and VI. Genetic differentiation was significant between each population except that between populations bordering Myanmar where transmigration was common. Regression analysis of pairwise linearized Fst and geographic distance suggests that P. vivax populations in Thailand have been isolated by distance. Sequence diversity of PvTRAP seems to be temporally stable over one decade in Tak province based on comparison of isolates collected in 1996 (n = 36) and 2006–2007. Besides natural selection, evidences of intragenic recombination have been supported in this study that could maintain and further generate diversity in this locus. It remains to be investigated whether amino acid substitutions in PvTRAP could influence host immune responses although several predicted variant T cell epitopes drastically altered the epitope scores. Knowledge on geographic diversity in PvTRAP constitutes an important basis for vaccine design provided that vaccination largely confers variant-specific immunity.

Malaria vaccine candidate antigen targeting the pre-erythrocytic stage of Plasmodium falciparum produced at high level in plants.

Plants have emerged as low-cost production platforms suitable for vaccines targeting poverty-related diseases. Besides functional efficacy, the stability, yield, and purification process determine the production costs of a vaccine and thereby the feasibility of plant-based production. We describe high-level plant production and functional characterization of a malaria vaccine candidate targeting the pre-erythrocytic stage of Plasmodium falciparum. CCT, a fusion protein composed of three sporozoite antigens (P. falciparum cell traversal protein for ookinetes and sporozoites [PfCelTOS], P. falciparum circumsporozoite protein [PfCSP], and P. falciparum thrombospondin-related adhesive protein [PfTRAP]), was transiently expressed by agroinfiltration in Nicotiana benthamiana leaves, accumulated to levels up to 2 mg/g fresh leaf weight (FLW), was thermostable up to 80°C and could be purified to >95% using a simple two-step procedure. Reactivity of sera from malaria semi-immune donors indicated the immunogenic conformation of the purified fusion protein consisting of PfCelTOS, PfCSP_TSR, PfTRAP_TSR domains (CCT) protein. Total IgG from the CCT-specific mouse immune sera specifically recognized P. falciparum sporozoites in immunofluorescence assays and induced up to 35% inhibition in hepatocyte invasion assays. Featuring domains from three promising sporozoite antigens with different roles (attachment and cell traversal) in the hepatocyte invasion process, CCT has the potential to elicit broader immune responses against the pre-erythrocytic stage of P. falciparum and represents an interesting new candidate, also as a component of multi-stage, multi-subunit malaria vaccine cocktails.

Immunogenicity of ChAd63 + MVA ME-TRAP in Senegalese adults

Background While malaria transmission is falling in many areas due to increased use of bed-nets and anti-malarials, it remains an enormous burden [1]. The leading malaria vaccine candidate, RTS,S, results in often considerably less than 60% efficacy against clinical malaria [2] resulting in a need for other approaches that may enhance this partial protection. One such approach is the use of viral vectors in a heterologous prime-boost regime with the aim of inducing a T-cell response. Previous studies have shown high levels of immunogenicity by using a chimpanzee adenovirus (ChAd63) followed by an attenuated vaccinia virus (modified vaccinia Ankara (MVA)) to deliver the pre-erythrocytic antigen, multiple epitope string with thrombospondin-related adhesion protein (ME-TRAP) [3].

Development of a Plasmodium vivax malaria vaccine for clinical applications using transgenic parasites, virus-like particles and recombinant viruses

As plans for malaria eradication continue, a vaccine targeting Plasmodium vivax is of major importance due to the fact that it is the most widely distributed human malaria parasite in the world and the most difficult to eliminate due to its ability to hide in the liver forming hypnozoites. Malaria liver-stage vaccines are one of the leading strategies and the only approach that has demonstrated complete, sterile protection in clinical trials. We have developed vaccine candidates using platforms suitable for human use, consisting of virus-like particles (VLPs), chimpanzee adenovirus (ChAd63) and modified vaccinia Ankara (MVA) containing malaria pre-erythrocytic antigens, and several novel challenge models using transgenic mouse-malaria P. berghei parasites expressing P. vivax antigens that permit the assessment of vaccine efficacy in small animal models, thus providing a suitable platform for vaccine development in non-endemic countries and permitting the progress of vaccines that rely on assessment of efficacy rather than measurement of immunogenicity, in the absence of a conclusive correlate of protection. We present results of two leading malaria vaccine candidates, a universal vivax circumsporozoite antigen (PvCSP) and the thrombospondin-related adhesion protein (PvTRAP) to create recombinant adenovirus, MVA and a platform consisting of VLPs to enhance efficacy of our malaria vaccines. All have been tested using transgenic parasites expressing the P. vivax CSP VK210, VK247 and TRAP, thus permitting the assessment of protective efficacy in various mouse strains and yielding important information to address cross protection against CSP using a challenge with VK210 or VK247 parasites in mice vaccinated with homologous, heterologous or chimeric CSP antigens. Our studies indicate that our vaccines are able to induce outstanding T-cell responses and antibody titers that can protect against a stringent challenge with high doses of fully virulent transgenic parasites in mice. Our groups are currently committing a substantial effort in developing further transgenic parasites to allow the discovery of new vaccine candidates able to afford protection.