MB2 is a novel sporozoite surface protein, that is present at several stages of the parasite lifecycle albeit in different sub-cellular locations as described earlier . It is composed of three distinct domains, an amino terminal basic domain, a central acidic domain and a carboxyl terminal GTP binding domain as shown in the schematic Figure 1A. The surface localization of MB2 at the sporozoite stage provides a potential target for an antibody mediated response. Within the MB2 basic domain three overlapping polypeptides, and one non-overlapping polypeptide spanning between the basic and acidic domains, and three more polypeptides covering the remainder of the acidic domain and a C-terminal polypeptide were initially generated as GST-His-tagged sandwich fusions and used to immunize rabbits. The immune rabbit IgG was used in an inhibition of sporozoite invasion (ISI) assay. Antibodies against MB2 B inhibited sporozoite invasion (57%), whilst antibodies directed against MB-FA and MB2-C inhibited poorly (33% and 18% respectively) as shown in Table 4, suggesting that antibodies directed against this non-repeat region may be important in protection against infection. All five protected volunteers produced anti-MB2 antibodies preferentially against the non-repeat MB2-B region, and the ISI assay showed that rabbit anti-MB2-B antibodies are more effective than antibodies against other regions of MB2 at blocking sporozoite invasion of hepatocytes. It may be speculated that anti-MB2-B antibodies may contribute to the overall protective sterile immunity acquired by non-replicating metabolically-active sporozoite immunized volunteers. Immunoblot analyses of plasma from individuals in an area of highly seasonal transmission during a period of high transmission also demonstrated higher frequencies of anti-MB2-B antibodies among individuals who were not infected with P. falciparum as compared to those who were. However, these individuals also had higher levels of anti-MB2-C antibodies. In this preliminary limited study the sera were obtained at a single point in time and we cannot know whether either type of antibodies were associated with protection from infection in this population. It is very likely that the inoculum of infection in these individuals was much lower than that of the irradiated sporozoite-infected individuals. The data suggest that individuals with intermittent natural P. falciparum exposure have B cell responses that include regions of the MB-2 antigen recognized by those protected individuals exposed to irradiated sporozoites. The qualitative and quantitative antibody responses to the area most strongly associated with protection in immunized volunteers (MB-2B) may be impaired in individuals in areas of seasonal transmission. There was no clear relationship between malaria symptoms and presence of antibodies to a specific region (MB-2B or MB-2C), but since presence of antibodies was assessed at the time of disease, we can conclude only that symptomatic and asymptomatic individuals in this area are able to mount antibody responses to MB-2B and MB-2C. Further studies are required to characterize these responses in individuals who reside in areas of high-level, year-round P. falciparum transmission, and prospective studies required to assess the association of these antibodies with protection from infection and disease in endemic areas.
One of the striking observations is that protected volunteers were able to recognize preferentially the non-repeat MB2-B and to a lesser degree the repeat-including MB2-C and -D regions that most naturally exposed persons in malaria-endemic areas recognize. The significance of such a qualitative difference in the antibody response is difficult to assess due to the limited sample size. It is known that at the optimal radiation dosage required to induce sterile immunity, the weakened sporozoite is not able to develop completely in the hepatocytes [30, 31]. Since the stage-dependent cellular localization of MB2 is accompanied by differential proteolytic processing , it may be that MB2 is aberrantly processed and is misdirected onto the surface of infected cells. Alternatively, B-cell response may be due to the exposure 'dose' of sporozoites. Protected volunteers received hundreds to more than a thousand bites of infected and irradiated mosquitoes in order to acquire sterile immunity . In contrast, in malaria endemic countries, exposed individuals receive on average less than 200 infective bites per year , and in the highland area of Kenya where the endemic serum samples were collected, the number of infective bites is likely to be much lower than this . It is possible that, the quantitative difference of inoculated sporozoites is responsible for the difference observed in antibody recognition against MB2 between protected volunteers and persons living in endemic countries. However antibodies against MB2-B were more frequent in those without parasitaemia than those with parasitaemia in the malaria endemic area, consistent with the findings in the protected versus unprotected volunteers.
The antigenic regions in the basic domain of MB2 were analyzed for potential amino acid polymorphisms. The nucleotide sequence of the MB2 gene was determined for field isolates obtained from different malaria-endemic regions of the world. It was expected that samples derived from non-overlapping locales would provide the greatest opportunity to detect sequence variation in MB2. The analysis of the primary structure of amplified MB2 DNA fragments from different isolates of P. falciparum showed that antigenic variation is unlikely to be a factor contributing to the different antibody response against MB2. Except for variation in the number of repeat units, the antigenic region in the B domain of MB2 is absolutely conserved among laboratory strains and field isolates collected from different parts of the world as shown in Figure 2. The amino acid sequence conservation may reflect a functional constraint of the B domain since it is not only exposed on the surface of the sporozoite but also translocated into the nucleus of blood-stage parasites. In our in vitro ISI study, antibodies to this conserved, antigenic region of MB2 can inhibit sporozoite invasion of hepatocytes. If these antibodies play a role in protective immunity in vivo, the finding that the antigenic region of MB2 is highly conserved suggests that it might make a good target for immune attack by antibodies since most, if not all, P. falciparum sporozoites would be recognized.
The Plasmodium parasite is genetically complex, and based on the malaria genome sequencing projects  may have 5,000-6,000 genes. Its antigenic composition also is expected to be complex. Thus, the challenge in designing effective malaria vaccine(s) is to define the immunogenic molecules that are essential and the methods to present them properly to the immune system to induce the desired immune responses that protected the volunteers experimentally immunized with the non-replicating metabolically-active sporozoite vaccine. The MB2 protein possesses a number of molecular and immunogenic properties that indicate it is an intriguing candidate to complement current vaccine studies. Studies have shown that immunity to malaria also is mediated, at least partly, by cellular immune mechanisms . In endemic areas, cytotoxic T lymphocytes (CTLs) from exposed individuals recognize epitopes in a number of pre-erythrocytic antigens of P. falciparum, and indirect evidence indicates that these CTLs may play a role in protective immunity [36–38]. Since MB2 was shown to be present in multiple developmental stages including the hepatic stage that can be recognized by CTLs, it will be important to obtain evidence that MB2 also is recognized by CTLs from P. falciparum -exposed individuals. The variation in the nature and strength of immune response observed in the endemic plasma samples may also be due to host factors such as MHC class I and II restriction. Further studies across multiple populations are needed to assess the type and strength of responses in individuals of differing genetic backgrounds. It is anticipated that as more novel parasite immunogens are characterized, the knowledge gained from studying them will help bridge the gap between recombinant and attenuated sporozoite vaccines.
The P. falciparum antigen MB2 is a multi-domain sporozoite surface protein . In this study denatured recombinant peptide fragments MB2 were probed with immune plasma or sera resulting in the detection of antibodies directed against linear epitopes. Immunoblot analyses using serum of a volunteer protected by the exposure to non-replicating metabolically active sporozoites (#5) revealed that linear epitopes within the basic domain of MB2 are recognized strongly, whilst the acidic domain is recognized poorly. In comparison, plasma from a person living in an endemic region (KU 162) contained antibodies against linear epitopes within both basic and acidic domains. Antibodies against linear epitopes within the GTP binding domain were not detected in either set of samples. Moreover, sera from all the protected volunteers (non-replicating, metabolically-active sporozoite immunized) contain antibodies against MB2. In contrast, no anti-MB2 antibodies were detected in sera of non-protected (also non-replicating metabolically-active sporozoite immunized) volunteers. Furthermore, antibodies from the serum of protected volunteers recognized preferentially the non-repeat-containing MB2-B peptide, while the repeat-containing MB2-C peptide is recognized preferentially by the antibodies in the plasma of persons living in malaria-endemic areas. In addition, although the acidic domain also contains two amino acid repeat regions , serum from a protected volunteer showed minimal antibody reactivity against the acidic domain. In contrast, two regions of the acidic domain, one of which contains amino acid repeats, were recognized strongly by the antibodies in the endemic plasma (MB2-D & MB2-E). These qualitative results are interpreted to indicate that there may be one or more B-cell epitopes encoded in the non-repeat regions of MB2-B, that are more relevant to protective immunity than those encoded in the repeat regions. The results of the inhibition of sporozoite invasion assay suggest that antibodies directed against epitopes encoded in the non-repeat region (MB2-B) possess greater anti-parasitic activities than antibodies against epitopes encoded in the repeat-included region (MB2-C). The in vitro ISI assay showed that anti-MB2-B antibodies are more effective than anti-MB2-FA and anti-MB2-C antibodies at blocking sporozoites from invading the hepatocyte. Although ~40% of the parasites are still able to enter HepG2 cells, it is not known whether their intra-hepatic development is affected. The change of the cellular location of MB2 from the surface to the nucleus as the parasite lifecycle progresses from the sporozoite stage to the erythrocytic stage is consistent with the interpretation that it may have a function in the development of the parasite . Thus, it is possible that anti-MB2 antibodies, although they partially block invasion of the sporozoites, they could also hinder the intra-hepatic development.