3D Analysis of the TCR/pMHCII Complex Formation in Monkeys Vaccinated with the First Peptide Inducing Sterilizing Immunity against Human Malaria

T-cell receptor gene rearrangements were studied in Aotus monkeys developing high antibody titers and sterilizing immunity against the Plasmodium falciparum malaria parasite upon vaccination with the modified synthetic peptide 24112, which was identified in the Merozoite Surface Protein 2 (MSP-2) and is known to bind to HLA-DR beta 1*0403 molecules with high capacity. Spectratyping analysis showed a preferential usage of V beta 12 and V beta 6 TCR gene families in 67% of HLA-DR beta 1*0403-like genotyped monkeys. Docking of peptide 24112 into the HLA-DR beta 1*0401-HA peptide-HA1.7TCR complex containing the VDJ rearrangements identified in fully protected monkeys showed a different structural signature compared to nonprotected monkeys. These striking results show the exquisite specificity of the TCR/pMHCII complex formation needed for inducing sterilizing immunity and provide important hints for a logical and rational methodology to develop multiepitopic, minimal subunit-based synthetic vaccines against infectious diseases, among them malaria.

Using the PfEMP1 Head Structure Binding Motif to Deal a Blow at Severe Malaria

Plasmodium falciparum (Pf) malaria causes 200 million cases worldwide, 8 million being severe and complicated leading to similar to 1 million deaths and similar to 100,000 abortions annually. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) has been implicated in cytoadherence and infected erythrocyte rosette formation, associated with cerebral malaria; chondroitin sulphate-A attachment and infected erythrocyte sequestration related to pregnancy-associated malaria and other severe forms of disease. An endothelial cell high activity binding peptide is described in several of this similar to 300 kDa hypervariable protein's domains displaying a conserved motif (GACxPxRRxxLC); it established H-bonds with other binding peptides to mediate red blood cell group A and chondroitin sulphate attachment. This motif (when properly modified) induced PfEMP1-specific strain-transcending, fully-protective immunity for the first time in experimental challenge in Aotus monkeys, opening the way forward for a long sought-after vaccine against severe malaria.

Identification of plasmodium vivax proteins with potential role in invasion using sequence redundancy reduction and profile hidden markov models

Background: This study describes a bioinformatics approach designed to identify Plasmodium vivax proteins potentially involved in reticulocyte invasion. Specifically, different protein training sets were built and tuned based on different biological parameters, such as experimental evidence of secretion and/or involvement in invasion-related processes. A profile-based sequence method supported by hidden Markov models (HMMs) was then used to build classifiers to search for biologically-related proteins. The transcriptional profile of the P. vivax intra-erythrocyte developmental cycle was then screened using these classifiers. Results: A bioinformatics methodology for identifying potentially secreted P. vivax proteins was designed using sequence redundancy reduction and probabilistic profiles. This methodology led to identifying a set of 45 proteins that are potentially secreted during the P. vivax intra-erythrocyte development cycle and could be involved in cell invasion. Thirteen of the 45 proteins have already been described as vaccine candidates; there is experimental evidence of protein expression for 7 of the 32 remaining ones, while no previous studies of expression, function or immunology have been carried out for the additional 25. Conclusions: The results support the idea that probabilistic techniques like profile HMMs improve similarity searches. Also, different adjustments such as sequence redundancy reduction using Pisces or Cd-Hit allowed data clustering based on rational reproducible measurements. This kind of approach for selecting proteins with specific functions is highly important for supporting large-scale analyses that could aid in the identification of genes encoding potential new target antigens for vaccine development and drug design. The present study has led to targeting 32 proteins for further testing regarding their ability to induce protective immune responses against P. vivax malaria.

New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin

The present study evaluated the immunogenicity of new malaria vaccine formulations based on the 19 kDa C-terminal fragment of Plasmodium vivax Merozoite Surface Protein-1 (MSP1(19)) and the Salmonella enterica serovar Typhimurium flagellin (FIiC), a Toll-like receptor 5 (TLR5) agonist. FHC was used as an adjuvant either admixed or genetically linked to the P. vivax MSP1(19) and administered to C57BL/6 mice via parenteral (s.c.) or mucosal (i.n.) routes. The recombinant fusion protein preserved MSP1(19) epitopes recognized by Sera collected from P. vivax infected humans and TLR5 agonist activity. Mice parenterally immunized with recombinant P vivax MSPI 19 in the presence of FliC, either admixed or genetically linked, elicited strong and long-lasting MSP1 (19)-specific systemic antibody responses with a prevailing IgG1 subclass response. Incorporation of another TLR agonist, CpG ODN 1826, resulted in a more balanced response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response measured by interferon-gamma secretion. Finally, we show that MSPI 19-specific antibodies recognized the native protein expressed on the surface of P. vivax parasites harvested from infected humans. The present report proposes a new class of malaria vaccine formulation based on the use of malaria antigens and the innate immunity agonist FliC. it contains intrinsic adjuvant properties and enhanced ability to induce specific humoral and cellular immune responses when administered alone or in combination with other adjuvants. (C) 2008 Elsevier Ltd. All rights reserved.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coil and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His6FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund`s adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity. (c) 2010 Elsevier Ltd. All rights reserved.

Geographic differentiation of polymorphism in the Plasmodium falciparum malaria vaccine candidate gene SERA5

SERA5 is regarded as a promising malaria vaccine candidate of the most virulent human malaria parasite Plasmodium falciparum. SERA5 is a 120 kDa abundantly expressed blood-stage protein containing a papain-like protease. Since substantial polymorphism in blood-stage vaccine candidates may potentially limit their efficacy, it is imperative to fully investigate polymorphism of the SERA5 gene (sera5). In this study, we performed evolutionary and population genetic analysis of sera5. The level of inter-species divergence (kS = 0.076) between P. falciparum and Plasmodium reichenowi, a closely related chimpanzee malaria parasite is comparable to that of housekeeping protein genes. A signature of purifying selection was detected in the proenzyme and enzyme domains. Analysis of 445 near full-length P. falciparum sera5 sequences from nine countries in Africa, Southeast Asia, Oceania and South America revealed extensive variations in the number of octamer repeat (OR) and serine repeat (SR) regions as well as substantial level of single nucleotide polymorphism (SNP) in non-repeat regions (2562 bp). Remarkably, a 14 amino acid sequence of SERA5 (amino acids 59-72) that is known to be the in vitro target of parasite growth inhibitory antibodies was found to be perfectly conserved in all 445 worldwide isolates of P. falciparum evaluated. Unlike other major vaccine target antigen genes such as merozoite surface protein-1, apical membrane antigen-1 or circumsporozoite protein, no strong evidence for positive selection was detected for SNPs in the non-repeat regions of sera5. A biased geographical distribution was observed in SNPs as well as in the haplotypes of the sera5 OR and SR regions. In Africa, OR- and SR-haplotypes with low frequency (<5%) and SNPs with minor allele frequency (<5%) were abundant and were mostly continent-specific. Consistently, significant genetic differentiation, assessed by the Wright's fixation index (FST) of inter-population variance in allele frequencies, was detected for SNPs and both OR- and SR-haplotypes among almost all parasite populations. The exception was parasite populations between Tanzania and Ghana, suggesting frequent gene flow in Africa. The present study points to the importance of investigating whether biased geographical distribution for SNPs and repeat variants in the OR and SR regions affect the reactivity of human serum antibodies to variants. (C) 2011 Elsevier Ltd. All rights reserved.

Estudio conformacional de péptidos modificados de las proteínas STARP, CelTOS, TRSP y SERA 5 de Plasmodium falciparum y su relación con una respuesta inmune en malaria

El desarrollo de una vacuna contra malaria es un área de exploración activa pero con enormes retos debido especialmente a la complejidad del ciclo del parásito. Así, es necesario bloquear las diferentes etapas de la invasión que tiene el Plasmodium falciparum y extraer de ellas la mayor información posible de la artillería que utiliza para su ataque. Para esto, péptidos de las proteínas STARP, CelTOS y TRSP (del esporozoito) y SERA 5 (del merozoito) que tienen alta afinidad de unión a células HepG2 y a glóbulos rojos respectivamente (conocidos como cHABPs), han sido modificados (conocidos como mHABPs), sintetizados y evaluados a nivel de respuesta inmune en monos Aotus así como estudiados en su conformación estructural por RMN de 1H. Los resultados muestran que los péptidos nativos no son inmunogénicos, pero pueden inducir altos títulos de anticuerpos cuando sus residuos críticos o sus vecinos son reemplazados por otro con un volumen y masa similar, pero diferente polaridad. El estudio conformacional pone de manifiesto que las estructuras de los péptidos nativos son diferentes de sus péptidos modificados ya sea que muestren regiones estructuradas más cortas o más largas o que no presenten ninguna, en comparación con sus análogos modificados altamente inmunogénicos. Las características estereoquímicas particulares en las cadenas laterales de algunos residuos de aminoácidos de estos péptidos modificados así como los rasgos fisicoquímicos parecen jugar un rol importante en la respuesta inmune apropiada cuando estos fueron inmunizados en grupos de monos Aotus confiriendo un avance al diseño de una vacuna contra malaria totalmente eficaz.

Qualitative analysis of preputial and vaginal bacterial microbiota in owl monkeys (Aotus azarai infulatus) raised in captivity

Background: The aim of this study was to identify the aerobic bacteria of the preputial and vaginal microbiota in owl monkeys that have been raised in captivity and to evaluate the antimicrobial susceptibility of these bacteria by gender and social organization. Methods: Thirty clinically healthy Aotus azarai infulatus were used. A total of 134 samples were collected, 60 from the preputial mucosa and 74 from the vaginal mucosa. An automated system of bacterial identification was used. Results and Conclusions: Staphylococcus intermedius and Proteus mirabilis were the microorganisms that were most frequently identified according to gender and social organization. The antimicrobial susceptibility of the isolated gram-positive bacteria was similar in both sexes. However, the gram-negative strains had some differences. The aerobic bacterial population of the vaginal and preputial microbiota is similar in owl monkeys, and there are no differences in the number and bacterial species according to sex and social organization. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Analysis of follicular events in owl monkeys (Aotus azarai infulatus) using B-mode and Doppler ultrasound

Ultrasound (B-mode) was used to analyze follicular events in 12 trained female owl monkeys (Aotus azarai infulatus). The animals were examined every 48 hours for over 90days to measure and map follicular growth in both ovaries and to measure (using Doppler velocimetry) local hemodynamic changes during the peri-ovulatory stage. There were 44 follicular growth events, each with two or three follicular waves, and a mean ± SEM interval between events of 17 ± 1.13 days. There were various hemodynamic changes during follicular growth; both vascular resistance index and pulsatility index decreased during the time when the follicle diameter peaked. Thus, both B-mode and Doppler ultrasound were useful for monitoring ovarian follicular events in owl monkeys. © 2013 Elsevier Inc.

"Identification and Diversity of Killer Cell Ig-Like Receptors in Aotus vociferans, a New World Monkey "

Previous BAC clone analysis of the Platyrrhini owl monkey KIRs have shown an unusual genetic structure in some loci. Therefore, cDNAs encoding KIR molecules from eleven Aotus vociferans monkeys were characterized here; tenputative KIR loci were found, some of which encoded atypical proteins such as KIR4DL and transcripts predicted to encode a D0+D1 configuration (AOTVOKIR2DL1*01v1) which appear to be unique in the Aotus genus. Furthermore, alternative splicing was found as a likely mechanism for producing activator receptors in A. vociferans species. KIR proteins from New World monkeys may be split into three new lineages according to domain by domain phylogenetic analysis. Although the A. vociferans KIR family displayed a high divergence among paralogous genes, individual loci were limited in their genetic polymorphism. Selection analysis showed that both constrained and rapid evolution may operate within the AvKIR family. The frequent alternative splicing (as a likely mechanism generating activator receptors), the presence of KIR4DL and KIR2DL1 (D0+D1) molecules and other data reported here suggest that the KIR family in Aotus has had a rapid evolution, independent from its Catarrhini counterparts.from its Catarrhini counterparts.

Uterine evaluation and gestation diagnosis in owl monkey (Aotus azarai infulatus) using the B mode ultrasound

Background Gynecological and obstetrical ultrasonography has become an indispensable tool in the routine management, health evaluation and research on captive non-human primates.Methods Ultrasound was used to evaluate the uterus and estimate the gestation of owl monkeys. Twelve couples were selected, where five were primiparous and seven multiparous females from the National Primate Center reproductive colony, Ananindeua-PA, Brazil. The procedures were carried out using the GE (R) Logiq 100 MP, equipped with a 7.5 MHz linear probe.Results the females showed a simple uterus, of elongated shape, regular outline and homogeneous echogenic texture. In the uterine measurements craniocaudal diameter, dorsoventral diameter and uterine volume (UV), significant differences were identified (P < 0.05) between ultrasound examinations of primiparous and multiparous females. The UV showed a positive correlation with the number of births. The gestational sac and the embryonic echo were visible between 28 and 38 days after mating. Between 48 and 68 days after mating, embryonic death was identified in all the gestations.Conclusions the chemical (use of tranquilizers) and husbandry factors (capture stress) may be related to the prenatal death. The establishing methods of conditioning the female to the ultrasonographic exam may offer a solution to this problem.

Renal Evaluation of Aotus azarai infulatus by Ultrasonography and Serum Chemistry Profile

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ovarian and Uterine Ultrasonography in Aotus azarai infulatus

We evaluated the uterus and ovaries of owl monkeys (Aotus azarai infulatus) via gynecological ultrasound examination. We evaluated the subjects in 2 different time periods. The first period (P1) was characterized by the absence of mating, with daily examinations, during 4 mo (n = 10). At the end of P1, we paired the subjects for 30 d, but without ultrasonographic evaluation. The second period (P2) was characterized by the presence of mating, with examinations once a week, during 7 consecutive months (n = 9). We evaluated the uterus and ovaries in sagittal and transverse scans, using a 5-12 MHz linear array probe. The uterine volume (UV) was directly proportional to the number of previous parturitions. The right ovary volume (RtOV) is greater than the left (LtOV) in P1 and P2. There is a positive correlation (p < 0.05) between the females' mass, RtOV (r = 0.28) and LtOV (r = 0.16).

Hematological, hepatic, and renal evaluation in Aotus azarai infulatus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effect of age and number of parturitions on uterine and ovarian variables in owl monkeys

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)