Showcasing Environmental Health and Safety Activities During the Coronavirus Disease 2019 Pandemic.

Introduction: Emergency preparedness is not a novel topic. What has been novel is the fast pace at which organizations, including academic institutions, have had to adapt to infectious disease outbreaks since 2000. Objective: The goal of this article is to highlight the various environmental health and safety (EHS) team activities during the coronavirus disease 2019 (COVID-19) pandemic to ensure that on-site personnel was safe, the research could be conducted, and critical business operations such as academics, laboratory animal care, environmental compliance, and routine healthcare functions could continue during the pandemic. Methods: The response framework is presented by discussing first the lessons learned in preparedness and emergency response during outbreaks that occurred since 2000, namely Influenza virus, Zika virus, and Ebola virus. Then, how the response to the COVID-19 pandemic was activated, and the effects of ramping down research and business activities. Results: Next, the contributions of each EHS unit are presented, namely, environmental, industrial hygiene and occupational safety, research safety and biosafety, radiation safety, supporting healthcare activities, disinfection, and communications and training. Discussion: Lastly, a few lessons learned are shared with the reader for moving toward normalcy.

MaHPIC malaria systems biology data from Plasmodium cynomolgi sporozoite longitudinal infections in macaques.

Plasmodium cynomolgi causes zoonotic malarial infections in Southeast Asia and this parasite species is important as a model for Plasmodium vivax and Plasmodium ovale. Each of these species produces hypnozoites in the liver, which can cause relapsing infections in the blood. Here we present methods and data generated from iterative longitudinal systems biology infection experiments designed and performed by the Malaria Host-Pathogen Interaction Center (MaHPIC) to delve deeper into the biology, pathogenesis, and immune responses of P. cynomolgi in the Macaca mulatta host. Infections were initiated by sporozoite inoculation. Blood and bone marrow samples were collected at defined timepoints for biological and computational experiments and integrative analyses revolving around primary illness, relapse illness, and subsequent disease and immune response patterns. Parasitological, clinical, haematological, immune response, and -omic datasets (transcriptomics, proteomics, metabolomics, and lipidomics) including metadata and computational results have been deposited in public repositories. The scope and depth of these datasets are unprecedented in studies of malaria, and they are projected to be a F.A.I.R., reliable data resource for decades.

Considerations for Laboratory Biosafety and Biosecurity During the Coronavirus Disease 2019 Pandemic: Applying the ISO 35001:2019 Standard and High-Reliability Organizations Principles.

Background: Risk assessment is a critical tool for evaluating emerging pathogens such as severe acute respiratory syndrome coronavirus 2 because of the limited available information about pathogens and the diseases they cause. Industries adopt unique frameworks for risk assessment, for example, the ISO 35001:2019 biorisk management for laboratories and other related organizations provide tools to identify, assess, control, and monitor risks associated with hazardous biological materials. Industries such as aerospace are known as high-reliability organizations (HROs) because these must balance high-risk operations with minimal catastrophic outcomes. HROs focus on five core principles: preoccupation with failure, reluctance to simplify, sensitivity to operations, resilience, and deference to expertise to evaluate and manage risk. Results: In the present discussion, practices described in the ISO 35001 standard and the HRO model are applied to the current challenges faced by laboratories worldwide. Laboratories processing known or unknown coronavirus disease 2019 (COVID-19) samples, testing COVID-19 vaccine candidates, propagating severe acute respiratory syndrome-associated coronavirus-2, or validating diagnostic assays benefit from implementing such practices. Principles extrapolated from the HRO also help illustrate the importance of the end-to-end processes to ensure successful outcomes. Summary: Workplace safety is enhanced by the involvement of all stakeholders, from top leadership to front-line workers. High-quality outcomes as measured by a lack of incidents, accidents, injuries, or near misses are the positive consequences of strictly following standard operating procedures and timely communication of risks and pitfalls. Adopting a systematic framework to identify and manage risks posed by emerging pathogens results in increased workplace safety and higher quality processes and products.

A Section on Service Animals in the Microbiology Teaching Laboratory Has Been Included in the 2019 Update to the Guidelines for Biosafety in Teaching Laboratories.

The accommodation of service animals in microbiology teaching labs has been included in the 2019 update to the American Society of Microbiology (ASM) Guidelines for Safety in Microbiology Laboratories. This commentary includes a legal framework related to service animals, the elements included in the 2019 ASM update, and additional risk-assessment considerations for the biosafety professional.

Distinct amino acid and lipid perturbations characterize acute versus chronic malaria.

Chronic malaria is a major public health problem and significant challenge for disease eradication efforts. Despite its importance, the biological factors underpinning chronic malaria are not fully understood. Recent studies have shown that host metabolic state can influence malaria pathogenesis and transmission, but its role in chronicity is not known. Here, with the goal of identifying distinct modifications in the metabolite profiles of acute versus chronic malaria, metabolomics was performed on plasma from Plasmodium-infected humans and nonhuman primates with a range of parasitemias and clinical signs. In rhesus macaques infected with Plasmodium coatneyi, significant alterations in amines, carnitines, and lipids were detected during a high parasitemic acute phase and many of these reverted to baseline levels once a low parasitemic chronic phase was established. Plasmodium gene expression, studied in parallel in the macaques, revealed transcriptional changes in amine, fatty acid, lipid and energy metabolism genes, as well as variant antigen genes. Furthermore, a common set of amines, carnitines, and lipids distinguished acute from chronic malaria in plasma from human Plasmodium falciparum cases. In summary, distinct host-parasite metabolic environments have been uncovered that characterize acute versus chronic malaria, providing insights into the underlying host-parasite biology of malaria disease progression.

Plasmodium vivax Parasite Load Is Associated With Histopathology in Saimiri boliviensis With Findings Comparable to P vivax Pathogenesis in Humans.

BACKGROUND: Plasmodium vivax can cause severe malaria with multisystem organ dysfunction and death. Clinical reports suggest that parasite accumulation in tissues may contribute to pathogenesis and disease severity, but direct evidence is scarce. METHODS: We present quantitative parasitological and histopathological analyses of tissue sections from a cohort of naive, mostly splenectomized Saimiri boliviensis infected with P vivax to define the relationship of tissue parasite load and histopathology. RESULTS: The lung, liver, and kidney showed the most tissue injury, with pathological presentations similar to observations reported from autopsies. Parasite loads correlated with the degree of histopathologic changes in the lung and liver tissues. In contrast, kidney damage was not associated directly with parasite load but with the presence of hemozoin, an inflammatory parasite byproduct. CONCLUSIONS: This analysis supports the use of the S boliviensis infection model for performing detailed histopathological studies to better understand and potentially design interventions to treat serious clinical manifestations caused by P vivax.

NIH Guidelines April 2019.

Following the required review period initiated in August 2018, the Department of Health and Human Services, National Institutes of Health (NIH), published the final changes to the Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) in April 2019. This amendment focused on the review, registration, and reporting requirements for human gene transfer studies. In addition, the Recombinant DNA Advisory Committee (RAC) was renamed the Novel and Exceptional Technology and Research Advisory Committee (NeXTRAC) to serve as an advisory body on emerging technologies.

Foreword and Introduction to This Issue on Contemporary Safety Topics in Animal Research.

Institutions with animal care and use programs are obligated to provide for the health and well-being of the animals, but are equally obligated to provide for safety of individuals associated with the program. The topics in this issue of the ILAR Journal, in association with those within the complimentary issue of the Journal of Applied Biosafety, provide a variety of contemporary occupational health and safety considerations in today's animal research programs. Each article addresses key or emerging occupational health and safety topics in institutional animal care and use programs, where the status of the topic, contemporary challenges, and future directions are provided.

Plasmodium cynomolgi infections in rhesus macaques display clinical and parasitological features pertinent to modelling vivax malaria pathology and relapse infections.

BACKGROUND: Plasmodium vivax infections in humans or in new world monkeys pose research challenges that necessitate the use of alternative model systems. Plasmodium cynomolgi is a closely related species that shares genetic and biological characteristics with P. vivax, including relapses. Here, the haematological dynamics and clinical presentation of sporozoite-initiated P. cynomolgi infections in Macaca mulatta (rhesus macaques) are evaluated over a 100-day period. METHODS: Five M. mulatta were inoculated with 2000 P. cynomolgi B strain sporozoites. Parasitological and haematological data were collected daily to study the clinical presentations of primary infections and relapses. Peripheral blood and bone marrow aspirates were collected at specific time points during infection for future and retrospective systems biology analyses. RESULTS: Patent infections were observed between days 10 and 12, and the acute, primary infection consisted of parasitaemias ranging from 269,962 to 1,214,842 parasites/µl (4.42-19.5 % parasitaemia). All animals presented with anaemia, ranging from moderate (7-10 g/dl) to severe (<7 g/dl), based on peripheral haemoglobin concentrations. Minimum haemoglobin levels coincided with the clearance of parasites and peripheral reticulocytosis was evident at this time. Mild thrombocytopaenia (<150,000 platelets/µl) was observed in all animals, but unlike haemoglobin, platelets were lowest whenever peripheral parasitaemia peaked. The animals' conditions were classified as non-severe, severe or lethal (in one case) based upon their clinical presentation. The lethal phenotype presented uniquely with an exceptionally high parasitaemia (19.5 %) and lack of a modest reticulocyte release, which was observed in the other animals prior to acute manifestations. One or two relapses were observed in the four surviving animals, and these were characterized by significantly lower parasitaemias and minimal changes in clinical parameters compared to pre-infection values. CONCLUSIONS: Rhesus macaque infections initiated by P. cynomolgi B strain sporozoites recapitulated pathology of human malaria, including anaemia and thrombocytopaenia, with inter-individual differences in disease severity. Importantly, this study provides an in-depth assessment of clinical and parasitological data, and shows that unlike the primary infections, the relapses did not cause clinical malaria. Notably, this body of research has provided experimental plans, large accessible datasets, and blood and bone marrow samples pertinent for ongoing and iterative systems biology investigations.

In silico Identification and Validation of a Linear and Naturally Immunogenic B-Cell Epitope of the Plasmodium vivax Malaria Vaccine Candidate Merozoite Surface Protein-9.

Synthetic peptide vaccines provide the advantages of safety, stability and low cost. The success of this approach is highly dependent on efficient epitope identification and synthetic strategies for efficacious delivery. In malaria, the Merozoite Surface Protein-9 of Plasmodium vivax (PvMSP9) has been considered a vaccine candidate based on the evidence that specific antibodies were able to inhibit merozoite invasion and recombinant proteins were highly immunogenic in mice and humans. However the identities of linear B-cell epitopes within PvMSP9 as targets of functional antibodies remain undefined. We used several publicly-available algorithms for in silico analyses and prediction of relevant B cell epitopes within PMSP9. We show that the tandem repeat sequence EAAPENAEPVHENA (PvMSP9E795-A808) present at the C-terminal region is a promising target for antibodies, given its high combined score to be a linear epitope and located in a putative intrinsically unstructured region of the native protein. To confirm the predictive value of the computational approach, plasma samples from 545 naturally exposed individuals were screened for IgG reactivity against the recombinant PvMSP9-RIRII729-972 and a synthetic peptide representing the predicted B cell epitope PvMSP9E795-A808. 316 individuals (58%) were responders to the full repetitive region PvMSP9-RIRII, of which 177 (56%) also presented total IgG reactivity against the synthetic peptide, confirming it validity as a B cell epitope. The reactivity indexes of anti-PvMSP9-RIRII and anti-PvMSP9E795-A808 antibodies were correlated. Interestingly, a potential role in the acquisition of protective immunity was associated with the linear epitope, since the IgG1 subclass against PvMSP9E795-A808 was the prevalent subclass and this directly correlated with time elapsed since the last malaria episode; however this was not observed in the antibody responses against the full PvMSP9-RIRII. In conclusion, our findings identified and experimentally confirmed the potential of PvMSP9E795-A808 as an immunogenic linear B cell epitope within the P. vivax malaria vaccine candidate PvMSP9 and support its inclusion in future subunit vaccines.

Plasmodium vivax trophozoite-stage proteomes.

Plasmodium vivax is the causative infectious agent of 80-300 million annual cases of malaria. Many aspects of this parasite's biology remain unknown. To further elucidate the interaction of P. vivax with its Saimiri boliviensis host, we obtained detailed proteomes of infected red blood cells, representing the trophozoite-enriched stage of development. Data from two of three biological replicate proteomes, emphasized here, were analyzed using five search engines, which enhanced identifications and resulted in the most comprehensive P. vivax proteomes to date, with 1375 P. vivax and 3209 S. boliviensis identified proteins. Ribosome subunit proteins were noted for both P. vivax and S. boliviensis, consistent with P. vivax's known reticulocyte host-cell specificity. A majority of the host and pathogen proteins identified belong to specific functional categories, and several parasite gene families, while 33% of the P. vivax proteins have no reported function. Hemoglobin was significantly oxidized in both proteomes, and additional protein oxidation and nitration was detected in one of the two proteomes. Detailed analyses of these post-translational modifications are presented. The proteins identified here significantly expand the known P. vivax proteome and complexity of available host protein functionality underlying the host-parasite interactive biology, and reveal unsuspected oxidative modifications that may impact protein function. BIOLOGICAL SIGNIFICANCE: Plasmodium vivax malaria is a serious neglected disease, causing an estimated 80 to 300 million cases annually in 95 countries. Infection can result in significant morbidity and possible death. P. vivax, unlike the much better-studied Plasmodium falciparum species, cannot be grown in long-term culture, has a dormant form in the liver called the hypnozoite stage, has a reticulocyte host-cell preference in the blood, and creates caveolae vesicle complexes at the surface of the infected reticulocyte membranes. Studies of stage-specific P. vivax expressed proteomes have been limited in scope and focused mainly on pathogen proteins, thus limiting understanding of the biology of this pathogen and its host interactions. Here three P. vivax proteomes are reported from biological replicates based on purified trophozoite-infected reticulocytes from different Saimiri boliviensis infections (the main non-human primate experimental model for P. vivax biology and pathogenesis). An in-depth analysis of two of the proteomes using 2D LC/MS/MS and multiple search engines identified 1375 pathogen proteins and 3209 host proteins. Numerous functional categories of both host and pathogen proteins were identified, including several known P. vivax protein family members (e.g., PHIST, eTRAMP and VIR), and 33% of protein identifications were classified as hypothetical. Ribosome subunit proteins were noted for both P. vivax and S. boliviensis, consistent with this parasite species' known reticulocyte host-cell specificity. In two biological replicates analyzed for post-translational modifications, hemoglobin was extensively oxidized, and various other proteins were also oxidized or nitrated in one of the two replicates. The cause of such protein modification remains to be determined but could include oxidized heme and oxygen radicals released from the infected red blood cell's parasite-induced acidic digestive vacuoles. In any case, the data suggests the presence of distinct infection-specific conditions whereby both the pathogen and host infected red blood cell proteins may be subject to significant oxidative stress.

Comparative transcriptomics and metabolomics in a rhesus macaque drug administration study.

We describe a multi-omic approach to understanding the effects that the anti-malarial drug pyrimethamine has on immune physiology in rhesus macaques (Macaca mulatta). Whole blood and bone marrow (BM) RNA-Seq and plasma metabolome profiles (each with over 15,000 features) have been generated for five naïve individuals at up to seven timepoints before, during and after three rounds of drug administration. Linear modeling and Bayesian network analyses are both considered, alongside investigations of the impact of statistical modeling strategies on biological inference. Individual macaques were found to be a major source of variance for both omic data types, and factoring individuals into subsequent modeling increases power to detect temporal effects. A major component of the whole blood transcriptome follows the BM with a time-delay, while other components of variation are unique to each compartment. We demonstrate that pyrimethamine administration does impact both compartments throughout the experiment, but very limited perturbation of transcript or metabolite abundance was observed following each round of drug exposure. New insights into the mode of action of the drug are presented in the context of pyrimethamine's predicted effect on suppression of cell division and metabolism in the immune system.

Naturally acquired immune responses to P. vivax merozoite surface protein 3α and merozoite surface protein 9 are associated with reduced risk of P. vivax malaria in young Papua New Guinean children.

BACKGROUND: Plasmodium vivax is the most geographically widespread human malaria parasite. Cohort studies in Papua New Guinea have identified a rapid onset of immunity against vivax-malaria in children living in highly endemic areas. Although numerous P. vivax merozoite antigens are targets of naturally acquired antibodies, the role of many of these antibodies in protective immunity is yet unknown. METHODOLOGY/PRINCIPAL FINDINGS: In a cohort of children aged 1-3 years, antibodies to different regions of Merozoite Surface Protein 3α (PvMSP3α) and Merozoite Surface Protein 9 (PvMSP9) were measured and related to prospective risk of P. vivax malaria during 16 months of active follow-up. Overall, there was a low prevalence of antibodies to PvMSP3α and PvMSP9 proteins (9-65%). Antibodies to the PvMSP3α N-terminal, Block I and Block II regions increased significantly with age while antibodies to the PvMSP3α Block I and PvMSP9 N-terminal regions were positively associated with concurrent P. vivax infection. Independent of exposure (defined as the number of genetically distinct blood-stage infection acquired over time (molFOB)) and age, antibodies specific to both PvMSP3α Block II (adjusted incidence ratio (aIRR) = 0.59, p = 0.011) and PvMSP9 N-terminus (aIRR = 0.68, p = 0.035) were associated with protection against clinical P. vivax malaria. This protection was most pronounced against high-density infections. For PvMSP3α Block II, the effect was stronger with higher levels of antibodies. CONCLUSIONS: These results indicate that PvMSP3α Block II and PvMSP9 N-terminus should be further investigated for their potential as P. vivax vaccine antigens. Controlling for molFOB assures that the observed associations are not confounded by individual differences in exposure.

Plasmodium vivax merozoite surface protein-3 (PvMSP3): expression of an 11 member multigene family in blood-stage parasites.

BACKGROUND: Three members of the Plasmodium vivax merozoite surface protein-3 (PvMSP3) family (PvMSP3-α, PvMSP3-ß and PvMSP3-γ) were initially characterized and later shown to be part of a larger highly diverse family, encoded by a cluster of genes arranged head-to-tail in chromosome 10. PvMSP3-α and PvMSP3-ß have become genetic markers in epidemiological studies, and are being evaluated as vaccine candidates. This research investigates the gene and protein expression of the entire family and pertinent implications. METHODOLOGY/PRINCIPAL FINDINGS: A 60 kb multigene locus from chromosome 10 in P. vivax (Salvador 1 strain) was studied to classify the number of pvmsp3 genes present, and compare their transcription, translation and protein localization patterns during blood-stage development. Eleven pvmsp3 paralogs encode an N-terminal NLRNG signature motif, a central domain containing repeated variable heptad sequences, and conserved hydrophilic C-terminal features. One additional ORF in the locus lacks these features and was excluded as a member of the family. Transcripts representing all eleven pvmsp3 genes were detected in trophozoite- and schizont-stage RNA. Quantitative immunoblots using schizont-stage extracts and antibodies specific for each PvMSP3 protein demonstrated that all but PvMSP3.11 could be detected. Homologs were also detected by immunoblot in the closely related simian species, P. cynomolgi and P. knowlesi. Immunofluorescence assays confirmed that eight of the PvMSP3s are present in mature schizonts. Uniquely, PvMSP3.7 was expressed exclusively at the apical end of merozoites. CONCLUSION/SIGNIFICANCE: Specific proteins were detected representing the expression of 10 out of 11 genes confirmed as members of the pvmsp3 family. Eight PvMSP3s were visualized surrounding merozoites. In contrast, PvMSP3.7 was detected at the apical end of the merozoites. Pvmsp3.11 transcripts were present, though no corresponding protein was detected. PvMSP3 functions remain unknown. The ten expressed PvMSP3s are predicted to have unique and complementary functions in merozoite biology.

Plasmodium vivax: modern strategies to study a persistent parasite's life cycle.

Plasmodium vivax has unique attributes to support its survival in varying ecologies and climates. These include hypnozoite forms in the liver, an invasion preference for reticulocytes, caveola-vesicle complex structures in the infected erythrocyte membrane and rapidly forming and circulating gametocytes. These characteristics make this species very different from P. falciparum. Plasmodium cynomolgi and other related simian species have identical biology and can serve as informative models of P. vivax infections. Plasmodium vivax and its model parasites can be grown in non-human primates (NHP), and in short-term ex vivo cultures. For P. vivax, in the absence of in vitro culture systems, these models remain highly relevant side by side with human clinical studies. While post-genomic technologies allow for greater exploration of P. vivax-infected blood samples from humans, these come with restrictions. Two advantages of NHP models are that infections can be experimentally tailored to address hypotheses, including genetic manipulation. Also, systems biology approaches can capitalise on computational biology combined with set experimental infection periods and protocols, which may include multiple sampling times, different types of samples, and the broad use of "omics" technologies. Opportunities for research on vivax malaria are increasing with the use of existing and new methodological strategies in combination with modern technologies.

Two functional reticulocyte binding-like (RBL) invasion ligands of zoonotic Plasmodium knowlesi exhibit differential adhesion to monkey and human erythrocytes.

BACKGROUND: Plasmodium knowlesi is a monkey malaria species that is becoming a serious public health concern infecting hundreds and perhaps thousands of humans in Southeast Asia. Invasion of erythrocytes by merozoites entails a cascade of molecular interactions. One step involves the adhesion of Plasmodium reticulocyte binding-like (RBL) proteins. Plasmodium knowlesi merozoites express only two RBL invasion ligands, known as Normocyte Binding Proteins (PkNBPXa and PkNBPXb). METHODS: Overlapping N-terminal regions of PkNBPXa and PkNBPXb were expressed in COS7 cells and tested for surface expression and adhesion to rhesus monkey erythrocytes. Subsequent tests to study specific receptor ligand interactions included adhesion to a panel of human and non-human primate erythrocytes, enzymatic treatment, and site directed mutagenesis. RESULTS: An N-terminal cysteine-rich region of PkNBPXb (PkNBPXb-II) exhibited specific adhesion to rhesus monkey erythrocytes. Mutation of four of five cysteines in PkNBPXb-II interfered with its surface expression on COS7 cells, suggesting disulphide bond conformation is critical for intracellular trafficking. Binding of PkNBPXb-II was abolished when rhesus erythrocytes were pre-treated with chymotrypsin, but not trypsin or neuraminidase. PkNBPXb-II also bound other Old World monkey species and gibbon erythrocytes. However, erythrocytes from other primate species including humans did not bind to PkNBPXb-II or native PkNBPXb. Importantly, unlike PkNBPXb, PkNBPXa bound human erythrocytes, and this binding was independent of the Duffy blood group determinant. CONCLUSIONS: The data reported here begins to clarify the functional domains of the P. knowlesi RBLs. A binding domain has been identified and characterized in PkNBPXb. Notably, this study demonstrates that unlike PkNBPXb, PkNBPXa can bind to human erythrocytes, suggesting that PkNBPXa may function as a ligand to enable the invasion of P. knowlesi merozoites into human cells.

Influence of HLA-DRB1 and HLA-DQB1 alleles on IgG antibody response to the P. vivax MSP-1, MSP-3α and MSP-9 in individuals from Brazilian endemic area.

BACKGROUND: The antibody response generated during malaria infections is of particular interest, since the production of specific IgG antibodies is required for acquisition of clinical immunity. However, variations in antibody responses could result from genetic polymorphism of the HLA class II genes. Given the increasing focus on the development of subunit vaccines, studies of the influence of class II alleles on the immune response in ethnically diverse populations is important, prior to the implementation of vaccine trials. METHODS AND FINDINGS: In this study, we evaluated the influence of HLA-DRB1* and -DQB1* allelic groups on the naturally acquired humoral response from Brazilian Amazon individuals (n = 276) against P. vivax Merozoite Surface Protein-1 (MSP-1), MSP-3α and MSP-9 recombinant proteins. Our results provide information concerning these three P. vivax antigens, relevant for their role as immunogenic surface proteins and vaccine candidates. Firstly, the studied population was heterogeneous presenting 13 HLA-DRB1* and 5 DQB1* allelic groups with a higher frequency of HLA-DRB1*04 and HLA-DQB1*03. The proteins studied were broadly immunogenic in a naturally exposed population with high frequency of IgG antibodies against PvMSP1-19 (86.7%), PvMSP-3 (77%) and PvMSP-9 (76%). Moreover, HLA-DRB1*04 and HLA-DQB1*03 alleles were associated with a higher frequency of IgG immune responses against five out of nine antigens tested, while HLA-DRB1*01 was associated with a high frequency of non-responders to repetitive regions of PvMSP-9, and the DRB1*16 allelic group with the low frequency of responders to PvMSP3 full length recombinant protein. CONCLUSIONS: HLA-DRB1*04 alleles were associated with high frequency of antibody responses to five out of nine recombinant proteins tested in Rondonia State, Brazil. These features could increase the success rate of future clinical trials based on these vaccine candidates.

A 95 kDa protein of Plasmodium vivax and P. cynomolgi visualized by three-dimensional tomography in the caveola-vesicle complexes (Schüffner's dots) of infected erythrocytes is a member of the PHIST family.

Plasmodium vivax and P. cynomolgi produce numerous caveola-vesicle complex (CVC) structures within the surface of the infected erythrocyte membrane. These contrast with the electron-dense knob protrusions expressed at the surface of Plasmodium falciparum-infected erythrocytes. Here we investigate the three-dimensional (3-D) structure of the CVCs and the identity of a predominantly expressed 95 kDa CVC protein. Liquid chromatography - tandem mass spectrometry analysis of immunoprecipitates by monoclonal antibodies from P. cynomolgi extracts identified this protein as a member of the Plasmodium helical interspersed subtelomeric (PHIST) superfamily with a calculated mass of 81 kDa. We named the orthologous proteins PvPHIST/CVC-81(95) and PcyPHIST/CVC-81(95) , analysed their structural features, including a PEXEL motif, repeated sequences and a C-terminal PHIST domain, and show that PHIST/CVC-81(95) is most highly expressed in trophozoites. We generated images of CVCs in 3-D using electron tomography (ET), and used immuno-ET to show PHIST/CVC-81(95) localizes to the cytoplasmic side of the CVC tubular extensions. Targeted gene disruptions were attempted in vivo. The pcyphist/cvc-81(95) gene was not disrupted, but parasites containing episomes with the tgdhfr selection cassette were retrieved by selection with pyrimethamine. This suggests that PHIST/CVC-81(95) is essential for survival of these malaria parasites.