Value Assessment Frameworks in Latin America: "Il buono, il brutto e il cattivo".

OBJECTIVES: Using a taxonomy previously developed, we assessed the strengths and limitations of available value assessment frameworks (VAF) in Latin America. METHODS: Systematic review of peer-reviewed journals, gray literature review, and surveys to ISPOR Latin America Industry Committee members were done to identify and select current VAF. Once selected, independent reviewers, organized by pairs, assessed each framework's input, methodology, and outputs. RESULTS: We assessed 7 of 9 VAF in the region, excluding 2 that were not currently in use. The review included 1 framework developed by a regional entity, and 6 country frameworks for either price assessment or to inform reimbursement. Most of these frameworks had a clear definition of the purpose (6 of 7) but could provide more details on the conceptual approach, including perspectives, methods for obtaining preferences, and the ability to incorporate multiple value dimensions (2 of 7). Most lacked information about inclusions/exclusions of elements included in the framework, and whether it assumes a base case comparator and how it is selected. The description of the evaluation of data sources and their scientific validity was inconsistently reported (3 of 7). Few included an assessment of the intervention's effect on total costs of treating a defined population (2 of 7), or a description of how uncertainty could be incorporated (3 of 7). Finally, potential conflicts of interest among those creating the framework are not sufficiently addressed (0 of 7). CONCLUSIONS: In the 7 frameworks assessed in Latin America, there are opportunities to improve dimensions, methods, and scope. Addressing these issues will strengthen these VAF for policy and clinical decision making.

Amino terminal peptides from the Plasmodium falciparum EBA-181/JESEBL protein bind specifically to erythrocytes and inhibit in vitro merozoite invasion.

Several EBA-175 paralogues (EBA-140, EBA-165, EBA-175, EBA-181, and EBL-1) have been described among the Plasmodium falciparum malaria parasite proteins, which are important in the red blood cell (RBC) invasion process. EBA-181/JESEBL is a 181 kDa protein expressed in the late schizont stage and located in the micronemes; it belongs to the Plasmodium Duffy binding-like family and is able to interact with the erythrocyte surface. Here, we describe the synthesis of 78, 20-mer synthetic peptides derived from the reported EBA-181/JESEBL sequence and their ability to bind RBCs in receptor-ligand assays. Five peptides (numbered 30030, 30031, 30045, 30051, and 30060) displayed high specific binding to erythrocytes; their equilibrium binding parameters were then determined. These peptides interacted with 53 and 33 kDa receptor proteins on the erythrocyte surface, this binding being altered when RBCs were pretreated with enzymes. They were able to inhibit P. falciparum merozoite invasion of RBCs when tested in in vitro assays. According to these results, these five EBA-181/JESEBL high specific erythrocyte binding peptides, as well as the entire protein, were seen to be involved in the molecular machinery used by the parasite for invading RBCs. They are thus suggested as potential candidates in designing a multi-sub-unit vaccine able to combat the P. falciparum malaria parasite.

Identifying Plasmodium falciparum merozoite surface protein-10 human erythrocyte specific binding regions.

Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine P. falciparum merozoite surface protein-10 (MSP-10) regions specifically binding to membrane surface receptors on human erythrocytes. Three MSP-10 protein High Activity Binding Peptides (HABPs) were identified, whose binding to erythrocytes became saturable and sensitive on being treated with neuraminidase, trypsin and chymotrypsin. Some of them specifically recognised a 50 kDa erythrocyte membrane protein. Some HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by 70%, suggesting that MSP-10 protein's possible role in the invasion process probably functions by using similar mechanisms to those described for other MSP family antigens. In addition to above results, the high homology in amino-acid sequence and superimposition of both MSP-10, MSP-8 and MSP-1 EGF-like domains and HABPs 31132, 26373 and 5501 suggest that tridimensional structure could be playing an important role in the invasion process and in designing synthetic multi-stage anti-malarial vaccines.

Identifying Plasmodium falciparum cytoadherence-linked asexual protein 3 (CLAG 3) sequences that specifically bind to C32 cells and erythrocytes.

Adhesion of mature asexual stage Plasmodium falciparum parasite-infected erythrocytes (iRBC) to the vascular endothelium is a critical event in the pathology of Plasmodium falciparum malaria. It has been suggested that the clag gene family is essential in cytoadherence to endothelial receptors. Primers used in PCR and RT-PCR assays allowed us to determine that the gene encoding CLAG 3 (GenBank accession no. NP_473155) is transcribed in the Plasmodium falciparum FCB2 strain. Western blot showed that antisera produced against polymerized synthetic peptides from this protein recognized a 142-kDa band in P. falciparum schizont lysate. Seventy-one 20-amino-acid-long nonoverlapping peptides, spanning the CLAG 3 (cytoadherence-linked asexual protein on chromosome 3) sequence were tested in C32 cell and erythrocyte binding assays. Twelve CLAG peptides specifically bound to C32 cells (which mainly express CD36) with high affinity, hereafter referred to as high-affinity binding peptides (HABPs). Five of them also bound to erythrocytes. HABP binding to C32 cells and erythrocytes was independent of peptide charge or peptide structure. Affinity constants were between 100 nM and 800 nM. Cross-linking and SDS-PAGE analysis allowed two erythrocyte binding proteins of around 26 kDa and 59 kDa to be identified, while proteins of around 53 kDa were identified as possible receptor sites for C-32 cells. The HABPs' role in Plasmodium falciparum invasion inhibition was determined. Such an approach analyzing various CLAG 3 regions may elucidate their functions and may help in the search for new antigens important for developing antimalarial vaccines.

Mapping the anatomy of a Plasmodium falciparum MSP-1 epitope using pseudopeptide-induced mono- and polyclonal antibodies and CD and NMR conformation analysis.

Antigen structure modulation represents an approach towards designing subunit malaria vaccines. A specific epitope's alpha carbon stereochemistry, as well as its backbone topochemistry, was assessed for obtaining novel malarial immunogens. A variety of MSP-1(38-61) Plasmodium falciparum epitope pseudopeptides derived were synthesised, based on solid-phase pseudopeptide chemistry strategies; these included all-L, all-D, partially-D substituted, all-Psi-[NH-CO]-Retro, all-Psi-[NH-CO]-Retro-inverso, and Psi-[CH2NH] reduced amide surrogates. We demonstrate that specific recombinant MSP-1(34-469) fragment binding to red blood cells (RBCs) is specifically inhibited by non-modified MSP-1(42-61), as well as by its V52-L53, M51-V52 reduced amide surrogates and partial-D substitutions in K48 and E49. In vivo tests revealed that reduced amide pseudopeptide-immunised Aotus monkeys induced neutralising antibodies specifically recognising the MSP-1 N-terminus region. These findings support the role of molecular conformation in malaria vaccine development.

Identifying Plasmodium falciparum EBA-175 homologue sequences that specifically bind to human erythrocytes.

Erythrocyte binding antigen-160 (EBA-160) protein is a Plasmodium falciparum antigen homologue from the erythrocyte binding protein family (EBP). It has been shown that the EBP family plays a role in parasite binding to the erythrocyte surface. The EBA-160 sequence has been chemically synthesised in seventy 20-mer sequential peptides covering the entire 3D7 protein strain, each of which was tested in erythrocyte binding assays to identify possible EBA-160 functional regions. Five EBA-160 high activity binding peptides (HABPs) specifically binding to erythrocytes with high affinity were identified. Dissociation constants lay between 200 and 460 nM and Hill coefficients between 1.5 and 2.3. Erythrocyte membrane protein binding peptide cross-linking assays using SDS-PAGE showed that these peptides bound specifically to 12, 28, and 44 kDa erythrocyte membrane proteins. The nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. HABPs were able to block merozoite in vitro invasion of erythrocytes. HABPs' potential as anti-malarial vaccine candidates is also discussed.

Specific erythrocyte binding capacity and biological activity of Plasmodium falciparum-derived rhoptry-associated protein 1 peptides.

Rhoptry-associated protein 1 (RAP1) is a merozoite antigen within Plasmodium falciparum rhoptries as yet having no specific function described for it. Synthetic peptides spanning the RAP1 sequence were tested in erythrocyte binding assays to identify possible RAP1 functional regions. Five high activity binding peptides (HABPs) were identified; 26201, 26202, 26203 and 26204 spanned residues 461C-K540 within RAP1 Cys region, whilst 26188 (201T-Y220) was located in p67 amino terminal. The results showed that peptide binding was saturable, some HABPs inhibited in vitro merozoite invasion and specifically bound to a 72 kDa protein in red blood cell membrane. HABP possible function in merozoite invasion of erythrocytes is also discussed.

Sporozoite and liver stage antigen Plasmodium falciparum peptides bind specifically to human hepatocytes.

Sporozoite and Liver Stage Antigen (SALSA) sequence synthetic peptides were used in HepG2 cell binding assays to identify regions involved in parasite invasion. SALSA 20608 ( 21IWASEKKDEKEASEQGEESHY40) and 20611 ( 64KKDDGTDKVQEKVLEKSPKY83) peptides were determined as having high binding activity in HepG2 cell assays, some of them were located in immunogenic regions. Immune-fluorescence antibody test with 24276 (20608 peptide analogue, CGIWSSMKMDEKMAAMQGEESHCG) showed sporozoite and merozoite reactivity. This data suggests SALSA high activity binding peptides' (HABPs) possible role in hepatic cell invasion and merozoite invasion of erythrocytes.

Identification of Plasmodium falciparum reticulocyte binding protein RBP-2 homologue a and b (PfRBP-2-Ha and -Hb) sequences that specifically bind to erythrocytes.

Plasmodium falciparum reticulocyte binding protein RBP-2 homologues a and b (PfRBP-2-Ha and -Hb) have been described as being high molecular weight proteins, expressed at the P. falciparum merozoite apical extreme, belonging to a family of proteins found in other Plasmodium involved in the search for erythrocyte populations before being invaded by merozoites. 185, 20-mer-long non-overlapping peptides, spanning the entire PfRBP-2-Ha and -Hb sequences, were synthesised, radiolabelled and tested in erythrocyte binding assays. Fifteen PfRBP-2-Ha and -Hb high binding activity peptides (HBAPs) specifically binding to erythrocytes with high affinity were identified. Dissociation constants were between 70 and 300 nM and Hill coefficients were 1 approximately. HBAPs residues critical for binding to erythrocytes were determined. Cross-linking was performed allowing possible receptors for PfRBP-2-Ha and -Hb to be identified on the surface of the erythrocytes. Some of the HABPs showed merozoite invasion inhibition greater than 90% in in vitro assays.

MAEBL Plasmodium falciparum protein peptides bind specifically to erythrocytes and inhibit in vitro merozoite invasion.

MAEBL is an erythrocyte binding protein located in the rhoptries and on the surface of mature merozoites, being expressed at the beginning of schizogony. The structure of MAEBL originally isolated from rodent malaria parasites suggested a molecule likely to be involved in invasion. We thus became interested in identifying possible MAEBL functional regions. Synthetic peptides spanning the MAEBL sequence were tested in erythrocyte binding assays to identify such possible MAEBL functional regions. Nine high activity binding peptides (HABPs) were identified: two were found in the M1 domain, one was found between the M1 and M2 regions, five in the erythrocyte binding domain (M2), and one in the protein's repeat region. The results showed that peptide binding was saturable; some HABPs inhibited in vitro merozoite invasion and specifically bound to a 33kDa protein on red blood cell membrane. HABPs' possible function in merozoite invasion of erythrocytes is also discussed.

P. falciparum: merozoite surface protein-8 peptides bind specifically to human erythrocytes.

This work determined Plasmodium falciparum merozoite surface protein-8 (MSP-8) regions specifically binding to membrane surface receptors on human erythrocytes. Five high activity binding peptides (HABPs), whose binding to erythrocytes became saturable and sensitive on being treated with neuraminidase and chymotrypsin were identified from the MSP-8 protein. Those amino acids directly involved in interaction with erythrocytes were also determined for each one of the HABPs. Some of them specifically recognized 28, 46, and 73 kDa erythrocyte membrane proteins. Some HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by up to 98%, suggesting the MSP-8 protein's possible role in the invasion process.

Peptides of the liver stage antigen-1 (LSA-1) of Plasmodium falciparum bind to human hepatocytes.

Synthetic peptides from the liver stage antigen-1 (LSA-1) antigen sequence were used in HepG2 cell and erythrocyte binding assays to identify regions that could be involved in parasite invasion. LSA-1 protein peptides 20630 ((21)INGKIIKNSEKDEIIKSNLRY(40)), 20637 ((157)KEKLQGQQSDSEQERRAY(173)), 20638 ((174)KEKLQEQQSDLEQERLAY(190)) and 20639 (191KEKLQEQQSDLEQERRAY(207)) had high binding activity in HepG2 assays. Were located in immunogenic regions; peptide cell binding was saturable. Peptide 20630 bound specifically to 48kDa HepG2 membrane surface protein. LSA-1 peptides 20630 ((21)INGKIIKNSEKDEIIKSNLRY(40)) and 20633 ((81)DKELTMSNVKNVSQTNFKSLY(100)) showed specific erythrocyte binding activity and inhibited merozoite invasion of erythrocytes in vitro. A monkey serum prepared against LSA-1 20630 peptide analog (CGINGKNIKNAEKPMIIKSNLRGC) inhibited merozoite invasion in vitro. The data suggest LSA-1 "High Activity Binding Peptides" could play a possible role in hepatic cell invasion as well as merozoite invasion of erythrocytes.