Non-variant specific antibody responses to the C-terminal region of merozoite surface protein-1 of Plasmodium falciparum (PfMSP-119) in Iranians exposed to unstable malaria transmission

<p>Abstract</p> <p>Background</p> <p>The C-terminal region of <it>Plasmodium falciparum </it>merozoite surface protein-1 (PfMSP-1₁₉) is a leading malaria vaccine candidate antigen. However, the existence of different variants of this antigen can limit efficacy of the vaccine development based on this protein. Therefore, in this study, the main objective was to define the frequency of PfMSP-1₁₉haplotypes in malaria hypoendemic region of Iran and also to analyse cross-reactive and/or variant-specific antibody responses to four PfMSP-1₁₉variant forms.</p> <p>Methods</p> <p>The PfMSP-1₁₉was genotyped in 50 infected subjects with <it>P. falciparum </it>collected during 2006-2008. Four GST-PfMSP-1₁₉variants (E/TSR/L, E/TSG/L, E/KNG/F and Q/KNG/L) were produced in <it>Escherichia coli </it>and naturally occurring IgG antibody to these proteins was evaluated in malaria patients' sera (n = 50) using ELISA. To determine the cross-reactivity of antibodies against each PfMSP-1₁₉variant in <it>P. falciparum-</it>infected human sera, an antibody depletion assay was performed in eleven corresponding patients' sera.</p> <p>Results</p> <p>Sequence data of the PfMSP-1₁₉revealed five variant forms in which the haplotypes Q/KNG/L and Q/KNG/F were predominant types and the second most frequent haplotype was E/KNG/F. In addition, the prevalence of IgG antibodies to all four PfMSP-1₁₉variant forms was equal and high (84%) among the studied patients' sera. Immunodepletion results showed that in Iranian malaria patients, Q/KNG/L variant could induce not only cross-reactive antibody responses to other PfMSP-1₁₉variants, but also could induce some specific antibodies that are not able to recognize the E/TSG/L or E/TSR/L variant forms.</p> <p>Conclusion</p> <p>The present findings demonstrated the presence of non-variant specific antibodies to PfMSP-1₁₉in Iranian falciparum malaria patients. This data suggests that polymorphism in PfMSP-1₁₉is less important and one variant of this antigen, particularly Q/KNG/L, may be sufficient to be included in PfMSP-1₁₉-based vaccine.</p

Cellular responses to modified Plasmodium falciparum MSP119 antigens in individuals previously exposed to natural malaria infection

<p>Abstract</p> <p>Background</p> <p>MSP1 processing-inhibitory antibodies bind to epitopes on the 19 kDa C-terminal region of the <it>Plasmodium falciparum </it>merozoite surface protein 1 (MSP1₁₉), inhibiting erythrocyte invasion. Blocking antibodies also bind to this antigen but prevent inhibitory antibodies binding, allowing invasion to proceed. Recombinant MSP1₁₉had been modified previously to allow inhibitory but not blocking antibodies to continue to bind. Immunization with these modified proteins, therefore, has the potential to induce more effective protective antibodies. However, it was unclear whether the modification of MSP1₁₉would affect critical T-cell responses to epitopes in this antigen.</p> <p>Methods</p> <p>The cellular responses to wild-type MSP1₁₉and a panel of modified MSP1₁₉antigens were measured using an <it>in-vitro </it>assay for two groups of individuals: the first were malaria-naïve and the second had been naturally exposed to <it>Plasmodium falciparum </it>infection. The cellular responses to the modified proteins were examined using cells from malaria-exposed infants and adults.</p> <p>Results</p> <p>Interestingly, stimulation indices (SI) for responses induced by some of the modified proteins were at least two-fold higher than those elicited by the wild-type MSP1₁₉. A protein with four amino acid substitutions (Glu27→Tyr, Leu31→Arg, Tyr34→Ser and Glu43→Leu) had the highest stimulation index (SI up to 360) and induced large responses in 64% of the samples that had significant cellular responses to the modified proteins.</p> <p>Conclusion</p> <p>This study suggests that specific MSP1₁₉variants that have been engineered to improve their antigenicity for inhibitory antibodies, retain T-cell epitopes and the ability to induce cellular responses. These proteins are candidates for the development of MSP1-based malaria vaccines.</p

Plasmodium berghei Circumvents Immune Responses Induced by Merozoite Surface Protein 1- and Apical Membrane Antigen 1-Based Vaccines

BACKGROUND: Two current leading malaria blood-stage vaccine candidate antigens for Plasmodium falciparum, the C-terminal region of merozoite surface protein 1 (MSP1(19)) and apical membrane antigen 1 (AMA1), have been prioritized because of outstanding protective efficacies achieved in a rodent malaria Plasmodium yoelii model. However, P. falciparum vaccines based on these antigens have had disappointing outcomes in clinical trials. Discrepancies in the vaccine efficacies observed between the P. yoelii model and human clinical trials still remain problematic. METHODOLOGY AND RESULTS: In this study, we assessed the protective efficacies of a series of MSP1(19)- and AMA1-based vaccines using the P. berghei rodent malarial parasite and its transgenic models. Immunization of mice with a baculoviral-based vaccine (BBV) expressing P. falciparum MSP1(19) induced high titers of PfMSP1(19)-specific antibodies that strongly reacted with P. falciparum blood-stage parasites. However, no protection was achieved following lethal challenge with transgenic P. berghei expressing PfMSP1(19) in place of native PbMSP1(19). Similarly, neither P. berghei MSP1(19)- nor AMA1-BBV was effective against P. berghei. In contrast, immunization with P. yoelii MSP1(19)- and AMA1-BBVs provided 100% and 40% protection, respectively, against P. yoelii lethal challenge. Mice that naturally acquired sterile immunity against P. berghei became cross-resistant to P. yoelii, but not vice versa. CONCLUSION: This is the first study to address blood-stage vaccine efficacies using both P. berghei and P. yoelii models at the same time. P. berghei completely circumvents immune responses induced by MSP1(19)- and AMA1-based vaccines, suggesting that P. berghei possesses additional molecules and/or mechanisms that circumvent the host's immune responses to MSP1(19) and AMA1, which are lacking in P. yoelii. Although it is not known whether P. falciparum shares these escape mechanisms with P. berghei, P. berghei and its transgenic models may have potential as useful tools for identifying and evaluating new blood-stage vaccine candidate antigens for P. falciparum

Formation of the Food Vacuole in Plasmodium falciparum: A Potential Role for the 19 kDa Fragment of Merozoite Surface Protein 1 (MSP119)

Plasmodium falciparum Merozoite Surface Protein 1 (MSP1) is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP119), which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP119 during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP119, fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP119 remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP119 and the chloroquine resistance transporter (CRT) as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP119 does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase

Protection Induced by Plasmodium falciparum MSP142 Is Strain-Specific, Antigen and Adjuvant Dependent, and Correlates with Antibody Responses

Vaccination with Plasmodium falciparum MSP142/complete Freund's adjuvant (FA) followed by MSP142/incomplete FA is the only known regimen that protects Aotus nancymaae monkeys against infection by erythrocytic stage malaria parasites. The role of adjuvant is not defined; however complete FA cannot be used in humans. In rodent models, immunity is strain-specific. We vaccinated Aotus monkeys with the FVO or 3D7 alleles of MSP142 expressed in Escherichia coli or with the FVO allele expressed in baculovirus (bv) combined with complete and incomplete FA, Montanide ISA-720 (ISA-720) or AS02A. Challenge with FVO strain P. falciparum showed that suppression of cumulative day 11 parasitemia was strain-specific and could be induced by E. coli expressed MSP142 in combination with FA or ISA-720 but not with AS02A. The coli42-FVO antigen induced a stronger protective effect than the bv42-FVO antigen, and FA induced a stronger protective effect than ISA-720. ELISA antibody (Ab) responses at day of challenge (DOC) were strain-specific and correlated inversely with c-day 11 parasitemia (r = −0.843). ELISA Ab levels at DOC meeting a titer of at least 115,000 ELISA Ab units identified the vaccinees not requiring treatment (noTx) with a true positive rate of 83.3% and false positive rate of 14.3 %. Correlation between functional growth inhibitory Ab levels (GIA) and cumulative day 11 parasitemia was weaker (r = −0.511), and was not as predictive for a response of noTx. The lowest false positive rate for GIA was 30% when requiring a true positive rate of 83.3%. These inhibition results along with those showing that antigen/FA combinations induced a stronger protective immunity than antigen/ISA-720 or antigen/AS02 combinations are consistent with protection as ascribed to MSP1-specific cytophilic antibodies. Development of an effective MSP142 vaccine against erythrocytic stage P. falciparum infection will depend not only on antigen quality, but also upon the selection of an optimal adjuvant component

Three-dimensional culture of human meniscal cells: Extracellular matrix and proteoglycan production

<p>Abstract</p> <p>Background</p> <p>The meniscus is a complex tissue whose cell biology has only recently begun to be explored. Published models rely upon initial culture in the presence of added growth factors. The aim of this study was to test a three-dimensional (3D) collagen sponge microenvironment (without added growth factors) for its ability to provide a microenvironment supportive for meniscal cell extracellular matrix (ECM) production, and to test the responsiveness of cells cultured in this manner to transforming growth factor-β (TGF-β).</p> <p>Methods</p> <p>Experimental studies were approved prospectively by the authors' Human Subjects Institutional Review Board. Human meniscal cells were isolated from surgical specimens, established in monolayer culture, seeded into a 3D scaffold, and cell morphology and extracellular matrix components (ECM) evaluated either under control condition or with addition of TGF-β. Outcome variables were evaluation of cultured cell morphology, quantitative measurement of total sulfated proteoglycan production, and immunohistochemical study of the ECM components chondroitin sulfate, keratan sulfate, and types I and II collagen.</p> <p>Result and Conclusion</p> <p>Meniscal cells attached well within the 3D microenvironment and expanded with culture time. The 3D microenvironment was permissive for production of chondroitin sulfate, types I and II collagen, and to a lesser degree keratan sulfate. This microenvironment was also permissive for growth factor responsiveness, as indicated by a significant increase in proteoglycan production when cells were exposed to TGF-β (2.48 μg/ml ± 1.00, mean ± S.D., vs control levels of 1.58 ± 0.79, p < 0.0001). Knowledge of how culture microenvironments influence meniscal cell ECM production is important; the collagen sponge culture methodology provides a useful in vitro tool for study of meniscal cell biology.</p

Breast cancer incidence highest in the range of one species of house mouse, Mus domesticus

Incidence of human breast cancer (HBC) varies geographically, but to date no environmental factor has explained this variation. Previously, we reported a 44% reduction in the incidence of breast cancer in women fully immunosuppressed following organ transplantation (Stewart et al (1995) Lancet346: 796–798). In mice infected with the mouse mammary tumour virus (MMTV), immunosuppression also reduces the incidence of mammary tumours. DNA with 95% identity to MMTV is detected in 40% of human breast tumours (Wang et al (1995) Cancer Res55: 5173–5179). These findings led us to ask whether the incidence of HBC could be correlated with the natural ranges of different species of wild mice. We found that the highest incidence of HBC worldwide occurs in lands where Mus domesticus is thse resident native or introduced species of house mouse. Given the similar responses of humans and mice to immunosuppression, the near identity between human and mouse MTV DNA sequences, and the close association between HBC incidence and mouse ranges, we propose that humans acquire MMTV from mice. This zoonotic theory for a mouse-viral cause of HBC allows testable predictions and has potential importance in prevention. © 2000 Cancer Research Campaig

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

Personalized recurrence risk assessment following the birth of a child with a pathogenic de novo mutation

Following the diagnosis of a paediatric disorder caused by an apparently de novo mutation, a recurrence risk of 1-2% is frequently quoted due to the possibility of parental germline mosaicism; but for any specific couple, this figure is usually incorrect. We present a systematic approach to providing individualized recurrence risk. By combining locus-specific sequencing of multiple tissues to detect occult mosaicism with long-read sequencing to determine the parent-of-origin of the mutation, we show that we can stratify the majority of couples into one of seven discrete categories associated with substantially different risks to future offspring. Among 58 families with a single affected offspring (representing 59 de novo mutations in 49 genes), the recurrence risk for 35 (59%) was decreased below 0.1%, but increased owing to parental mixed mosaicism for 5 (9%)-that could be quantified in semen for paternal cases (recurrence risks of 5.6-12.1%). Implementation of this strategy offers the prospect of driving a major transformation in the practice of genetic counselling