Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are efficient soluble intra... more Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are efficient soluble intracellular sensors that activate defense mechanisms against pathogens. In teleost fish, the involvement of NLRs in the immune response is not well understood. However, recent work has evidenced the expression of different NLRs in response to some pathogen associated molecular patterns (PAMPs). In the present work, the cDNA sequence encoding three new NOD-like receptors were identified in Oncorhynchus mykiss, namely OmNLRC3, OmNLRC5 and OmNLRX1. Results showed that their sequences coded for proteins of 1135, 836 and 1010 amino acids, respectively. The deduced protein sequences of all receptors showed characteristic domains of this receptor family, such as leucine rich repeats and NACHT domain. Phylogenetic analysis revealed a high degree of identity with other NOD-like receptors and they are clustered into different families. Transcript expression analysis indicated that OmNLRs are constitutively expressed in liver, spleen, intestine, gill, skin and brain. OmNLR expression was upregulated in kidney and gills from rainbow trout in response to LPS. In order to give new insights into the function of these new NLR members, an in vitro model of immune stimulation was established using the rainbow trout cell line RTgill-W1. Expression analysis revealed that RTgill-W1 overexpressed proinflammatory cytokines in response to LPS and poly I:C alongside with a differential overexpression of OmNLRC3, OmNLRC5 and OmNLRX1. The expression of OmNLRC5 was further verified at the protein level by immunofluorescence. Finally, the effect of the overexpressed cytokines on the OmNLR expression by RTgill-W1 cells was assessed, suggesting a regulatory mechanism on OmNLRC3 expression. Overall, results suggest that O. mykiss NOD-like receptors could play a key role in the defense mechanisms of teleost through PAMP recognition. Future studies will focus on gills which could be related with a key sensor mucosal system in one of the most environmentally fish exposed tissues.
Single dose immunisation is a major goal in vaccine design. The purpose of this study was the dev... more Single dose immunisation is a major goal in vaccine design. The purpose of this study was the development of a single dose delivery system for the SPf66 malaria vaccine, based on this antigen's microencapsulation in PLGA microspheres by double emulsion method. Results indicate that a single immunisation in mice and monkeys with the SPf66 malaria vaccine, encapsulated in a mixture of two formulations of PLGA microspheres, induced a remarkably high and long-lasting immune response as assessed by ELISA and Western Blott. This immune response was associated with a good protective capacity in Aotus monkeys, after experimental challenge, indicating that antigen integrity lasted following the microencapsulation process. PLGA biodegradable microspheres thus serve as an effective delivery system for the design of a single dose immunisation vaccine, such as the SPf66 synthetic malaria vaccine.
Background: Chile has achieved the highest coverage for vaccines against the SARS-CoV-2 virus wor... more Background: Chile has achieved the highest coverage for vaccines against the SARS-CoV-2 virus worldwide. Objective: To assess the progression of immunity (natural and acquired by vaccine) in a cohort from two Chilean cities. Methods: Individuals (n = 386) who participated in three phases of population-based serial prevalence studies were included (2020–2021 and 2022). Presence of SARS-CoV-2 antibodies was measured in serum. Data including time of vaccination and type of vaccine received were analysed with descriptive statistics. Results: Seroprevalence was 3.6% in the first round and increased to 96.9% in the second and 98.7% in the third. In the third round, 75% of individuals who had received the basal full scheme were seropositive at 180 days or more since their last dose; 98% of individuals who received one booster dose were seropositive at 180 days or more, and 100% participants who received two boosters were seropositive, regardless of time since their last dose. Participants ...
Using levels of neutralizing antibodies (nAbs), we evaluate the successful Chilean SARS-CoV-2 vac... more Using levels of neutralizing antibodies (nAbs), we evaluate the successful Chilean SARS-CoV-2 vaccine campaign, which combines technologies and heterologous boosters. In 120 randomly selected seropositive individuals from a population-based study, we conclude that the booster dose, regardless of vaccine technology or natural infection, and mRNA vaccines significantly improve nAbs response.
Objective: Chile is among the most successful nations worldwide in COVID-19 vaccine rollout. By D... more Objective: Chile is among the most successful nations worldwide in COVID-19 vaccine rollout. By December 31st, 2021, 84.1% of the population was fully vaccinated, and 56.1% received booster doses using DIFFERENT TYPES OF COVID-19 VACCINES. In this context, we aimed to estimate the anti-SARS-CoV-2 antibodies following the infection and vaccination campaign.Study design: Population-based cross-sectional serosurvey based on a representative sample of the cities of Santiago, Coquimbo/La Serena, and Talca used in a previous study.Methods: We selected the participants using a three-stage stratified sampling. They were blood-sampled on-site and answered a questionnaire regarding COVID-19-associated variables and vaccination antecedents using Wantai SARS-CoV-2 Ab ELISA to determine seroprevalence. This research followed the generic protocol of World Health Organization Unity studies.Results: We recruited 2,198 individuals aged 7-93 between October 5th and November 25th, 2021. In our sample,...
CoronaVac vaccine from Sinovac Life Science is currently being used in several countries. In Chil... more CoronaVac vaccine from Sinovac Life Science is currently being used in several countries. In Chile, the effectiveness of preventing hospitalization is higher than 80% with a vaccination schedule. However, to date, there are no data about immune response induction or specific memory. For this reason, we recruited 15 volunteers without previous suspected/diagnosed COVID-19 and with negative PCR over time to evaluate the immune response to CoronaVac 28 and 90 days after the second immunization (dpi). The CoronaVac administration induces total and neutralizing anti-spike antibodies in all vaccinated volunteers at 28 and 90 dpi. Furthermore, using ELISpot analysis to assay cellular immune responses against SARS-CoV-2 spike protein, we found an increase in IFN-gamma- and Granzyme B-producing cells in vaccinated volunteers at 28 and 90 dpi. Together, our results indicate that CoronaVac induces a robust humoral immune response and cellular immune memory of at least 90 dpi.
Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are efficient soluble intra... more Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are efficient soluble intracellular sensors that activate defense mechanisms against pathogens. In teleost fish, the involvement of NLRs in the immune response is not well understood. However, recent work has evidenced the expression of different NLRs in response to some pathogen associated molecular patterns (PAMPs). In the present work, the cDNA sequence encoding three new NOD-like receptors were identified in Oncorhynchus mykiss, namely OmNLRC3, OmNLRC5 and OmNLRX1. Results showed that their sequences coded for proteins of 1135, 836 and 1010 amino acids, respectively. The deduced protein sequences of all receptors showed characteristic domains of this receptor family, such as leucine rich repeats and NACHT domain. Phylogenetic analysis revealed a high degree of identity with other NOD-like receptors and they are clustered into different families. Transcript expression analysis indicated that OmNLRs are constitu...
Sporozoite and Liver Stage Antigen (SALSA) sequence synthetic peptides were used in HepG2 cell bi... more 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 (21 IWASEKKDEKEASEQGEESHY 40) and 20611 (64 KKDDGTDKVQEKVLEKSPKY 83) 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.
Virulence and immunity are still poorly understood in Mycobacterium tuberculosis. The H37Rv M. tu... more Virulence and immunity are still poorly understood in Mycobacterium tuberculosis. The H37Rv M. tuberculosis laboratory strain genome has been completely sequenced, and this along with proteomic technology represent powerful tools contributing toward studying the biology of target cell interaction with a facultative bacillus and designing new strategies for controlling tuberculosis. Rv2004c is a putative M. tuberculosis protein that could have specific mycobacterial functions. This study has revealed that the encoding gene is present in all mycobacterium species belonging to the M. tuberculosis complex. Rv2004c gene transcription was observed in all of this complex's strains except Mycobacterium bovis and Mycobacterium microti. Rv2004c protein expression was confirmed by using antibodies able to recognize a 54‐kDa molecule by immunoblotting, and its location was detected on the M. tuberculosis surface by transmission electron microscopy, suggesting that it is a mycobacterial surf...
The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium t... more The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT‐PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25‐kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20‐amino‐acid‐long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross‐linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.
Adhesion of mature asexual stage Plasmodium falciparum parasite-infected erythrocytes (iRBC) to t... more 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.
Synthetic peptides from the liver stage antigen-1 (LSA-1) antigen sequence were used in HepG2 cel... more 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 (191 KEKLQEQQSDLEQERRAY 207) had high binding activity in HepG2 assays. Were located in immunogenic regions; peptide cell binding was saturable. Peptide 20630 bound specifically to 48 kDa 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.
Human papillomaviruses (HPVs) are the cause of epithelial lesions, HPV type 16 and type 18 being ... more Human papillomaviruses (HPVs) are the cause of epithelial lesions, HPV type 16 and type 18 being associated with the development of anogenital cancer. The L1 Major Capsid Protein (L1) represents about 90% of total HPV protein and is involved in virus‐host cell interaction, but little is known about this binding process. L1 sequences from HPV types 16 and 18 were synthesized in 56 20‐mer peptides, covering the entire protein, HPLC‐purified, 125I‐radiolabeled and tested in VERO and HeLa cell‐binding assays to identify those peptides with high specific binding activity. Peptides 18283 (residues 54–77) and 18294 (274–308) from HPV16 L1, as well as 18312 (59–78) and 18322 (259–278) from HPV18 L1, presented high specific target cell binding activity. Peptide 18283 and 18294 affinity constants were 300 and 600 nM, respectively. Enzyme cell treatment before binding assay indicated that VERO and HeLa cell peptide receptor is a surface‐exposed protein. There was a 60% reduction in peptide 182...
Plasmodium falciparum malaria antigen is an integral membrane protein called apical membrane anti... more Plasmodium falciparum malaria antigen is an integral membrane protein called apical membrane antigen-1. High activity binding peptides to human red blood cells have been identi¢ed in this protein. 4337 is a conserved, non-immunogenic peptide with high activity red blood cell binding and its critical residues have already been identi¢ed. Peptide analogues (with amino acids having the same mass but di¡erent charge) were generated to change their immunogenic and protective characteristics. Three analogues having positive or negative immunological results were studied by nuclear magnetic resonance. The studied peptides all had an K K-helix fragment, but in di¡erent peptide regions and extensions, except for randomly structured 4337. We show that altering a few amino acids induced immunogenicity and protectivity against experimental malaria and changed their three-dimensional structure, suggesting a better ¢t with immune system molecules and that modi¢ed peptides having better immunological properties can be included in the design of new malaria multi-component subunit-based vaccine.
Plasmodium falciparum histoaspartic protease (HAP) is an active enzyme involved in haemoglobin de... more Plasmodium falciparum histoaspartic protease (HAP) is an active enzyme involved in haemoglobin degradation. HAP is expressed as an inactive 51-kDa zymogen and is cleaved into an active 37-kDa enzyme. It has been proposed that this kind of protease might be implicated in the parasite's invasion of erythrocytes; however, this protein's role during invasion has still to be determined. Synthetic peptides derived from the HAP precursor (proHAP) were tested in erythrocyte binding assays to identify their possible function in the invasion process. Two proHAP high-activity binding peptides (HABPs) specifically bound to erythrocytes; these peptides were numbered 30609 (101 LKNYIKESVKLFNKGLTKKS 120) and 30610 (121 YLG-SEFDNVELKDLANVLSF 140). The binding of these two peptides was saturable, presenting nanomolar affinity constants. These peptides interacted with 26-and 45-kDa proteins on the erythrocyte surface; the nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. The HABPs showed greater than 90% merozoite invasion inhibition in in vitro assays. Goat serum containing proHAP polymeric peptide antibodies inhibited parasite invasion in vitro.
Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studie... more 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
Biochemical and Biophysical Research Communications, 2004
Plasmodium vivax, one of the four parasite species causing malaria in humans, is the most widespr... more Plasmodium vivax, one of the four parasite species causing malaria in humans, is the most widespread throughout the world, leading to nearly 80 million cases per year, mainly in Latin-America and Asia. An open reading frame encoding the Plasmodium falciparum merozoite surface protein 8 P. vivax homologue has been identified in the present study by screening the current data obtained from this parasiteÕs partially sequenced genome. This new protein contains 487 amino-acids, two epidermal growth factor like domains, hydrophobic regions at the N-and C-termini compatible with a signal peptide, and a glycosylphosphatidylinositol anchor site, respectively. This geneÕs transcription and its encoded protein expression have been assessed, as well as its recognition by P. vivax-infected patientsÕ sera. Based on this recognition, and a previous study showing that mice immunised with the Plasmodium yoelii homologous protein were protected, we consider the PvMSP8 a good candidate to be included in a multi-stage multiantigen P. vivax vaccine.
Biochemical and Biophysical Research Communications, 2007
A hepatitis C virus E 2 protein-derived sequence was selected for studying the effect of N-glycos... more A hepatitis C virus E 2 protein-derived sequence was selected for studying the effect of N-glycosylation on the peptide chain's conformational structure. The results suggested that the 534 TDVF 537 motif contained in peptide 33402 (529 WGENDTDVFVLNNTRY 544) had a type III b-turn, relevant in antigen recognition of polyclonal antibodies, binding to human cells, and binding to HLA DRB1 * 0401 molecules. N-Glycopeptides derived from this sequence contained monosaccharides in Asn 532. N-Glycopeptides presented differences in peptide chain structure compared to non-glycosylated peptides. Peptide 33402 specifically bound to human cells, specificity becoming lost when it was N-glycosylated. N-Glycosylation decreased antigen recognition of mouse polyclonal sera against this sequence. N-Glycopeptide binding to HLA DRB1 * 0401 molecules was similar to that presented by non-glycosylated peptide, indicating that N-glycosylation did not affect binding to HLA DRB1 * 0401 molecules. N-Glycosylation induced changes at structural and functional level which could be relevant for modulating human cell binding properties and antibody recognition.
Biochemical and Biophysical Research Communications, 2005
The process of Mycobacterium tuberculosis infection of the macrophage implies a very little-known... more The process of Mycobacterium tuberculosis infection of the macrophage implies a very little-known initial recognition and adherence step, important for mycobacterial survival; many proteins even remain like hypothetical. The Rv1510c gene, encoding a putatively conserved membrane protein, was investigated by analysing the M. tuberculosis genome sequence data reported by Cole et al. and a previous report that used PCR assays to show that the Rv1510 gene was only present in M. tuberculosis. This article confirmed all the above and identified the transcribed gene in M. tuberculosis, Mycobacterium africanum, and in M. tuberculosis clinical isolates. Antibodies raised against peptides from this protein recognised a 44 kDa band, corresponding to Rv1510c theoretical mass (44,294 Da). Assays involving synthetic peptides covering the whole protein binding to U937 and A549 cell lines led to recognising five high activity binding peptides in the Rv1510 protein: 11094, 11095, 11105, 11108, and 11111. Their affinity constants and Hill coefficients were determined by using U937 cells. Cross-linking assays performed with some of these HABPs showed that they specifically bound to a U937 cell line 51 kDa protein, but not to Hep G2 or red blood cell proteins, showing this interactionÕs specificity.
Biochemical and Biophysical Research Communications, 2007
Four Plasmodium species cause malaria in humans, Plasmodium falciparum being the most widely stud... more Four Plasmodium species cause malaria in humans, Plasmodium falciparum being the most widely studied to date. All Plasmodium species have paired club-shaped organelles towards their apical extreme named rhoptries that contain many lipids and proteins which are released during target cell invasion. P. falciparum RhopH3 is a rhoptry protein triggering important immune responses in patients from endemic regions. It has also been shown that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes. Recent immunisation studies in mice with the Plasmodium yoelii and Plasmodium berghei RhopH3 P. falciparum homologue proteins found that they are able to induce protection in murine models. This study described identifying and characterising RhopH3 protein in Plasmodium vivax; it is encoded by a seven exon gene and expressed during the parasite's asexual stage. PvRhopH3 has similar processing to its homologue in P. falciparum and presents a cellular immunolocalisation pattern characteristic of rhoptry proteins.
Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are efficient soluble intra... more Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are efficient soluble intracellular sensors that activate defense mechanisms against pathogens. In teleost fish, the involvement of NLRs in the immune response is not well understood. However, recent work has evidenced the expression of different NLRs in response to some pathogen associated molecular patterns (PAMPs). In the present work, the cDNA sequence encoding three new NOD-like receptors were identified in Oncorhynchus mykiss, namely OmNLRC3, OmNLRC5 and OmNLRX1. Results showed that their sequences coded for proteins of 1135, 836 and 1010 amino acids, respectively. The deduced protein sequences of all receptors showed characteristic domains of this receptor family, such as leucine rich repeats and NACHT domain. Phylogenetic analysis revealed a high degree of identity with other NOD-like receptors and they are clustered into different families. Transcript expression analysis indicated that OmNLRs are constitutively expressed in liver, spleen, intestine, gill, skin and brain. OmNLR expression was upregulated in kidney and gills from rainbow trout in response to LPS. In order to give new insights into the function of these new NLR members, an in vitro model of immune stimulation was established using the rainbow trout cell line RTgill-W1. Expression analysis revealed that RTgill-W1 overexpressed proinflammatory cytokines in response to LPS and poly I:C alongside with a differential overexpression of OmNLRC3, OmNLRC5 and OmNLRX1. The expression of OmNLRC5 was further verified at the protein level by immunofluorescence. Finally, the effect of the overexpressed cytokines on the OmNLR expression by RTgill-W1 cells was assessed, suggesting a regulatory mechanism on OmNLRC3 expression. Overall, results suggest that O. mykiss NOD-like receptors could play a key role in the defense mechanisms of teleost through PAMP recognition. Future studies will focus on gills which could be related with a key sensor mucosal system in one of the most environmentally fish exposed tissues.
Single dose immunisation is a major goal in vaccine design. The purpose of this study was the dev... more Single dose immunisation is a major goal in vaccine design. The purpose of this study was the development of a single dose delivery system for the SPf66 malaria vaccine, based on this antigen's microencapsulation in PLGA microspheres by double emulsion method. Results indicate that a single immunisation in mice and monkeys with the SPf66 malaria vaccine, encapsulated in a mixture of two formulations of PLGA microspheres, induced a remarkably high and long-lasting immune response as assessed by ELISA and Western Blott. This immune response was associated with a good protective capacity in Aotus monkeys, after experimental challenge, indicating that antigen integrity lasted following the microencapsulation process. PLGA biodegradable microspheres thus serve as an effective delivery system for the design of a single dose immunisation vaccine, such as the SPf66 synthetic malaria vaccine.
Background: Chile has achieved the highest coverage for vaccines against the SARS-CoV-2 virus wor... more Background: Chile has achieved the highest coverage for vaccines against the SARS-CoV-2 virus worldwide. Objective: To assess the progression of immunity (natural and acquired by vaccine) in a cohort from two Chilean cities. Methods: Individuals (n = 386) who participated in three phases of population-based serial prevalence studies were included (2020–2021 and 2022). Presence of SARS-CoV-2 antibodies was measured in serum. Data including time of vaccination and type of vaccine received were analysed with descriptive statistics. Results: Seroprevalence was 3.6% in the first round and increased to 96.9% in the second and 98.7% in the third. In the third round, 75% of individuals who had received the basal full scheme were seropositive at 180 days or more since their last dose; 98% of individuals who received one booster dose were seropositive at 180 days or more, and 100% participants who received two boosters were seropositive, regardless of time since their last dose. Participants ...
Using levels of neutralizing antibodies (nAbs), we evaluate the successful Chilean SARS-CoV-2 vac... more Using levels of neutralizing antibodies (nAbs), we evaluate the successful Chilean SARS-CoV-2 vaccine campaign, which combines technologies and heterologous boosters. In 120 randomly selected seropositive individuals from a population-based study, we conclude that the booster dose, regardless of vaccine technology or natural infection, and mRNA vaccines significantly improve nAbs response.
Objective: Chile is among the most successful nations worldwide in COVID-19 vaccine rollout. By D... more Objective: Chile is among the most successful nations worldwide in COVID-19 vaccine rollout. By December 31st, 2021, 84.1% of the population was fully vaccinated, and 56.1% received booster doses using DIFFERENT TYPES OF COVID-19 VACCINES. In this context, we aimed to estimate the anti-SARS-CoV-2 antibodies following the infection and vaccination campaign.Study design: Population-based cross-sectional serosurvey based on a representative sample of the cities of Santiago, Coquimbo/La Serena, and Talca used in a previous study.Methods: We selected the participants using a three-stage stratified sampling. They were blood-sampled on-site and answered a questionnaire regarding COVID-19-associated variables and vaccination antecedents using Wantai SARS-CoV-2 Ab ELISA to determine seroprevalence. This research followed the generic protocol of World Health Organization Unity studies.Results: We recruited 2,198 individuals aged 7-93 between October 5th and November 25th, 2021. In our sample,...
CoronaVac vaccine from Sinovac Life Science is currently being used in several countries. In Chil... more CoronaVac vaccine from Sinovac Life Science is currently being used in several countries. In Chile, the effectiveness of preventing hospitalization is higher than 80% with a vaccination schedule. However, to date, there are no data about immune response induction or specific memory. For this reason, we recruited 15 volunteers without previous suspected/diagnosed COVID-19 and with negative PCR over time to evaluate the immune response to CoronaVac 28 and 90 days after the second immunization (dpi). The CoronaVac administration induces total and neutralizing anti-spike antibodies in all vaccinated volunteers at 28 and 90 dpi. Furthermore, using ELISpot analysis to assay cellular immune responses against SARS-CoV-2 spike protein, we found an increase in IFN-gamma- and Granzyme B-producing cells in vaccinated volunteers at 28 and 90 dpi. Together, our results indicate that CoronaVac induces a robust humoral immune response and cellular immune memory of at least 90 dpi.
Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are efficient soluble intra... more Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are efficient soluble intracellular sensors that activate defense mechanisms against pathogens. In teleost fish, the involvement of NLRs in the immune response is not well understood. However, recent work has evidenced the expression of different NLRs in response to some pathogen associated molecular patterns (PAMPs). In the present work, the cDNA sequence encoding three new NOD-like receptors were identified in Oncorhynchus mykiss, namely OmNLRC3, OmNLRC5 and OmNLRX1. Results showed that their sequences coded for proteins of 1135, 836 and 1010 amino acids, respectively. The deduced protein sequences of all receptors showed characteristic domains of this receptor family, such as leucine rich repeats and NACHT domain. Phylogenetic analysis revealed a high degree of identity with other NOD-like receptors and they are clustered into different families. Transcript expression analysis indicated that OmNLRs are constitu...
Sporozoite and Liver Stage Antigen (SALSA) sequence synthetic peptides were used in HepG2 cell bi... more 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 (21 IWASEKKDEKEASEQGEESHY 40) and 20611 (64 KKDDGTDKVQEKVLEKSPKY 83) 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.
Virulence and immunity are still poorly understood in Mycobacterium tuberculosis. The H37Rv M. tu... more Virulence and immunity are still poorly understood in Mycobacterium tuberculosis. The H37Rv M. tuberculosis laboratory strain genome has been completely sequenced, and this along with proteomic technology represent powerful tools contributing toward studying the biology of target cell interaction with a facultative bacillus and designing new strategies for controlling tuberculosis. Rv2004c is a putative M. tuberculosis protein that could have specific mycobacterial functions. This study has revealed that the encoding gene is present in all mycobacterium species belonging to the M. tuberculosis complex. Rv2004c gene transcription was observed in all of this complex's strains except Mycobacterium bovis and Mycobacterium microti. Rv2004c protein expression was confirmed by using antibodies able to recognize a 54‐kDa molecule by immunoblotting, and its location was detected on the M. tuberculosis surface by transmission electron microscopy, suggesting that it is a mycobacterial surf...
The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium t... more The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT‐PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25‐kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20‐amino‐acid‐long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross‐linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.
Adhesion of mature asexual stage Plasmodium falciparum parasite-infected erythrocytes (iRBC) to t... more 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.
Synthetic peptides from the liver stage antigen-1 (LSA-1) antigen sequence were used in HepG2 cel... more 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 (191 KEKLQEQQSDLEQERRAY 207) had high binding activity in HepG2 assays. Were located in immunogenic regions; peptide cell binding was saturable. Peptide 20630 bound specifically to 48 kDa 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.
Human papillomaviruses (HPVs) are the cause of epithelial lesions, HPV type 16 and type 18 being ... more Human papillomaviruses (HPVs) are the cause of epithelial lesions, HPV type 16 and type 18 being associated with the development of anogenital cancer. The L1 Major Capsid Protein (L1) represents about 90% of total HPV protein and is involved in virus‐host cell interaction, but little is known about this binding process. L1 sequences from HPV types 16 and 18 were synthesized in 56 20‐mer peptides, covering the entire protein, HPLC‐purified, 125I‐radiolabeled and tested in VERO and HeLa cell‐binding assays to identify those peptides with high specific binding activity. Peptides 18283 (residues 54–77) and 18294 (274–308) from HPV16 L1, as well as 18312 (59–78) and 18322 (259–278) from HPV18 L1, presented high specific target cell binding activity. Peptide 18283 and 18294 affinity constants were 300 and 600 nM, respectively. Enzyme cell treatment before binding assay indicated that VERO and HeLa cell peptide receptor is a surface‐exposed protein. There was a 60% reduction in peptide 182...
Plasmodium falciparum malaria antigen is an integral membrane protein called apical membrane anti... more Plasmodium falciparum malaria antigen is an integral membrane protein called apical membrane antigen-1. High activity binding peptides to human red blood cells have been identi¢ed in this protein. 4337 is a conserved, non-immunogenic peptide with high activity red blood cell binding and its critical residues have already been identi¢ed. Peptide analogues (with amino acids having the same mass but di¡erent charge) were generated to change their immunogenic and protective characteristics. Three analogues having positive or negative immunological results were studied by nuclear magnetic resonance. The studied peptides all had an K K-helix fragment, but in di¡erent peptide regions and extensions, except for randomly structured 4337. We show that altering a few amino acids induced immunogenicity and protectivity against experimental malaria and changed their three-dimensional structure, suggesting a better ¢t with immune system molecules and that modi¢ed peptides having better immunological properties can be included in the design of new malaria multi-component subunit-based vaccine.
Plasmodium falciparum histoaspartic protease (HAP) is an active enzyme involved in haemoglobin de... more Plasmodium falciparum histoaspartic protease (HAP) is an active enzyme involved in haemoglobin degradation. HAP is expressed as an inactive 51-kDa zymogen and is cleaved into an active 37-kDa enzyme. It has been proposed that this kind of protease might be implicated in the parasite's invasion of erythrocytes; however, this protein's role during invasion has still to be determined. Synthetic peptides derived from the HAP precursor (proHAP) were tested in erythrocyte binding assays to identify their possible function in the invasion process. Two proHAP high-activity binding peptides (HABPs) specifically bound to erythrocytes; these peptides were numbered 30609 (101 LKNYIKESVKLFNKGLTKKS 120) and 30610 (121 YLG-SEFDNVELKDLANVLSF 140). The binding of these two peptides was saturable, presenting nanomolar affinity constants. These peptides interacted with 26-and 45-kDa proteins on the erythrocyte surface; the nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. The HABPs showed greater than 90% merozoite invasion inhibition in in vitro assays. Goat serum containing proHAP polymeric peptide antibodies inhibited parasite invasion in vitro.
Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studie... more 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
Biochemical and Biophysical Research Communications, 2004
Plasmodium vivax, one of the four parasite species causing malaria in humans, is the most widespr... more Plasmodium vivax, one of the four parasite species causing malaria in humans, is the most widespread throughout the world, leading to nearly 80 million cases per year, mainly in Latin-America and Asia. An open reading frame encoding the Plasmodium falciparum merozoite surface protein 8 P. vivax homologue has been identified in the present study by screening the current data obtained from this parasiteÕs partially sequenced genome. This new protein contains 487 amino-acids, two epidermal growth factor like domains, hydrophobic regions at the N-and C-termini compatible with a signal peptide, and a glycosylphosphatidylinositol anchor site, respectively. This geneÕs transcription and its encoded protein expression have been assessed, as well as its recognition by P. vivax-infected patientsÕ sera. Based on this recognition, and a previous study showing that mice immunised with the Plasmodium yoelii homologous protein were protected, we consider the PvMSP8 a good candidate to be included in a multi-stage multiantigen P. vivax vaccine.
Biochemical and Biophysical Research Communications, 2007
A hepatitis C virus E 2 protein-derived sequence was selected for studying the effect of N-glycos... more A hepatitis C virus E 2 protein-derived sequence was selected for studying the effect of N-glycosylation on the peptide chain's conformational structure. The results suggested that the 534 TDVF 537 motif contained in peptide 33402 (529 WGENDTDVFVLNNTRY 544) had a type III b-turn, relevant in antigen recognition of polyclonal antibodies, binding to human cells, and binding to HLA DRB1 * 0401 molecules. N-Glycopeptides derived from this sequence contained monosaccharides in Asn 532. N-Glycopeptides presented differences in peptide chain structure compared to non-glycosylated peptides. Peptide 33402 specifically bound to human cells, specificity becoming lost when it was N-glycosylated. N-Glycosylation decreased antigen recognition of mouse polyclonal sera against this sequence. N-Glycopeptide binding to HLA DRB1 * 0401 molecules was similar to that presented by non-glycosylated peptide, indicating that N-glycosylation did not affect binding to HLA DRB1 * 0401 molecules. N-Glycosylation induced changes at structural and functional level which could be relevant for modulating human cell binding properties and antibody recognition.
Biochemical and Biophysical Research Communications, 2005
The process of Mycobacterium tuberculosis infection of the macrophage implies a very little-known... more The process of Mycobacterium tuberculosis infection of the macrophage implies a very little-known initial recognition and adherence step, important for mycobacterial survival; many proteins even remain like hypothetical. The Rv1510c gene, encoding a putatively conserved membrane protein, was investigated by analysing the M. tuberculosis genome sequence data reported by Cole et al. and a previous report that used PCR assays to show that the Rv1510 gene was only present in M. tuberculosis. This article confirmed all the above and identified the transcribed gene in M. tuberculosis, Mycobacterium africanum, and in M. tuberculosis clinical isolates. Antibodies raised against peptides from this protein recognised a 44 kDa band, corresponding to Rv1510c theoretical mass (44,294 Da). Assays involving synthetic peptides covering the whole protein binding to U937 and A549 cell lines led to recognising five high activity binding peptides in the Rv1510 protein: 11094, 11095, 11105, 11108, and 11111. Their affinity constants and Hill coefficients were determined by using U937 cells. Cross-linking assays performed with some of these HABPs showed that they specifically bound to a U937 cell line 51 kDa protein, but not to Hep G2 or red blood cell proteins, showing this interactionÕs specificity.
Biochemical and Biophysical Research Communications, 2007
Four Plasmodium species cause malaria in humans, Plasmodium falciparum being the most widely stud... more Four Plasmodium species cause malaria in humans, Plasmodium falciparum being the most widely studied to date. All Plasmodium species have paired club-shaped organelles towards their apical extreme named rhoptries that contain many lipids and proteins which are released during target cell invasion. P. falciparum RhopH3 is a rhoptry protein triggering important immune responses in patients from endemic regions. It has also been shown that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes. Recent immunisation studies in mice with the Plasmodium yoelii and Plasmodium berghei RhopH3 P. falciparum homologue proteins found that they are able to induce protection in murine models. This study described identifying and characterising RhopH3 protein in Plasmodium vivax; it is encoded by a seven exon gene and expressed during the parasite's asexual stage. PvRhopH3 has similar processing to its homologue in P. falciparum and presents a cellular immunolocalisation pattern characteristic of rhoptry proteins.
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Papers by Jimena Cortés