Susheel K Singh - Academia.edu (original) (raw)

Papers by Susheel K Singh

Research paper thumbnail of Antibody Responses Against Plasmodium falciparum MSP3 Protein During Natural Malaria Infection in Individuals Living in Malaria-Endemic Regions of India

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences

Research paper thumbnail of GMZ2 Vaccine-Induced Antibody Responses, Naturally Acquired Immunity and the Incidence of Malaria in Burkinabe Children

Frontiers in Immunology

GMZ2 is a malaria vaccine candidate evaluated in a phase 2b multi-centre trial. Here we assessed ... more GMZ2 is a malaria vaccine candidate evaluated in a phase 2b multi-centre trial. Here we assessed antibody responses and the association of naturally acquired immunity with incidence of malaria in one of the trial sites, Banfora in Burkina Faso. The analysis included 453 (GMZ2 = 230, rabies = 223) children aged 12-60 months old. Children were followed-up for clinical malaria episodes for 12 months after final vaccine administration. Antibody levels against GMZ2 and eleven non-GMZ2 antigens were measured on days 0 and 84 (one month after final vaccine dose). Vaccine efficacy (VE) differed by age group (interaction, (12-35 months compared to 36-60 months), p = 0.0615). During the twelve months of follow-up, VE was 1% (95% confidence interval [CI] -17%, 17%) and 23% ([CI] 3%, 40%) in the 12 - 35 and 36 – 60 months old children, respectively. In the GMZ2 group, day 84 anti-GMZ2 IgG levels were associated with reduced incidence of febrile malaria during the follow up periods of 1-6 months...

Research paper thumbnail of Peripheral Merozoite Surface Proteins Are Targets of Naturally Acquired Immunity against Malaria in both India and Ghana

Infection and Immunity, 2020

Development of a successful blood-stage vaccine against Plasmodium falciparum malaria remains a h... more Development of a successful blood-stage vaccine against Plasmodium falciparum malaria remains a high priority. Immune-epidemiological studies are effective tools for the identification of antigenic targets of naturally acquired immunity (NAI) against malaria. However, differences in study design and methodology may compromise interstudy comparisons.

Research paper thumbnail of Natural antibody responses to Plasmodium falciparum MSP3 and GLURP(R0) antigens are associated with low parasite densities in malaria patients living in the Central Region of Ghana

Parasites & Vectors, 2017

Research paper thumbnail of Naturally Acquired Antibodies Target the Glutamate-Rich Protein on Intact Merozoites and Predict Protection Against Febrile Malaria

The Journal of Infectious Diseases, 2017

Research paper thumbnail of Naturally acquired antibody response to Plasmodium falciparum describes heterogeneity in transmission on islands in Lake Victoria

Scientific Reports, 2017

As markers of exposure anti-malaria antibody responses can help characterise heterogeneity in mal... more As markers of exposure anti-malaria antibody responses can help characterise heterogeneity in malaria transmission. In the present study antibody responses to Plasmodium falciparum AMA-1, MSP-119 and CSP were measured with the aim to describe transmission patterns in meso-endemic settings in Lake Victoria. Two cross-sectional surveys were conducted in Lake Victoria in January and August 2012. The study area comprised of three settings: mainland (Ungoye), large island (Mfangano) and small islands (Takawiri, Kibuogi, Ngodhe). Individuals provided a finger-blood sample to assess malaria infection by microscopy and PCR. Antibody response to P. falciparum was determined in 4,112 individuals by ELISA using eluted dried blood from filter paper. The overall seroprevalence was 64.0% for AMA-1, 39.5% for MSP-119, and 12.9% for CSP. Between settings, seroprevalences for merozoite antigens were similar between Ungoye and Mfangano, but higher when compared to the small islands. For AMA-1, the se...

Research paper thumbnail of Heterologous Expression and Evaluation of Novel Plasmodium falciparum Transmission Blocking Vaccine Candidates

Frontiers in Immunology

Malaria transmission blocking vaccines (TBV) aim to induce antibodies that can interrupt Plasmodi... more Malaria transmission blocking vaccines (TBV) aim to induce antibodies that can interrupt Plasmodium falciparum development in the mosquito midgut and thereby prevent onward malaria transmission. A limited number of TBV candidates have been identified and only three (Pfs25, Pfs230 and Pfs48/45) have entered clinical testing. While one of these candidates may emerge as a highly potent TBV candidate, it is premature to determine if they will generate sufficiently potent and sustained responses. It is therefore important to explore novel candidate antigens. We recently analyzed sera from naturally exposed individuals and found that the presence and/or intensity of antibodies against 12 novel putative surface expressed gametocyte antigens was associated with transmission reducing activity. In this study, protein fragments of these novel TBV candidates were designed and heterologously expressed in Drosophila melanogaster S2 cells and Lactococcus lactis. Eleven protein fragments, covering ...

Research paper thumbnail of Additional file 3 of Antibody responses to a suite of novel serological markers for malaria surveillance demonstrate strong correlation with clinical and parasitological infection across seasons and transmission settings in The Gambia

Additional file 3. Map of household geolocation of PfAMA1 sero-positive, clinical malaria and Pf ... more Additional file 3. Map of household geolocation of PfAMA1 sero-positive, clinical malaria and Pf infections across four villages during dry and wet transmission seasons. Spatial distribution of infections shown for lower transmission (N'demban) and higher transmission (Besse) villages in the West Coast Region (WCR) and lower transmission (Njaiyal) and higher transmission (Madina Samako) villages in the Upper River Region (URR). Infections at the start of the wet season (June – July 2013) shown on the left and during the wet and transmission season (August – December 2013) on the right.

Research paper thumbnail of Method for Production of Cysteine-Rich Proteins in Lactococcus lactis Expression System

Methods in Molecular Biology, 2022

The Gram-positive bacterium Lactococcus lactis is an ideal expression host for the overproduction... more The Gram-positive bacterium Lactococcus lactis is an ideal expression host for the overproduction of heterologous proteins in a functional form. L. lactis has recently been identified as an efficient Gram-positive cell factory for the production of recombinant proteins and the safety of this production system has been confirmed in multiple clinical trials. Key desirable features of L. lactis include its generally recognized as safe (GRAS) status, long history of safe use in food production, probiotic properties, absence of endotoxins, capacity to secrete stable recombinant protein to the growth medium, the presence of few proteases, and a diverse selection of cloning and inducible expression vectors. Growth of lactococci is rapid, proceeds to high cell densities, and does not require aeration, which facilitates large-scale fermentation. We have previously described the production of several Plasmodium falciparum antigens with varying degrees of predicted structural complexities, those which are considered difficult-to-produce proteins by using L. lactis pH-dependent inducible promoter (P170). The purpose of this chapter is to provide a detailed protocol for the expression of difficult-to-produce proteins, mainly high cysteine-rich proteins, in the soluble form in L. lactis from cloning of the target gene to the determination of expression levels and purification.

Research paper thumbnail of Data from: Unravelling the immune signature of Plasmodium falciparum transmission-reducing immunity

Infection with Plasmodium can elicit antibodies that inhibit parasite survival in the mosquito, w... more Infection with Plasmodium can elicit antibodies that inhibit parasite survival in the mosquito, when they are ingested in an infectious blood meal. Here, we determine the transmission-reducing activity (TRA) of naturally acquired antibodies from 648 malaria-exposed individuals using lab-based mosquito-feeding assays. Transmission inhibition is significantly associated with antibody responses to Pfs48/45, Pfs230, and to 43 novel gametocyte proteins assessed by protein microarray. In field-based mosquito-feeding assays the likelihood and rate of mosquito infection are significantly lower for individuals reactive to Pfs48/45, Pfs230 or to combinations of the novel TRA-associated proteins. We also show that naturally acquired purified antibodies against key transmission-blocking epitopes of Pfs48/45 and Pfs230 are mechanistically involved in TRA, whereas sera depleted of these antibodies retain high-level, complement-independent TRA. Our analysis demonstrates that host antibody response...

Research paper thumbnail of Conserved cysteine residues in malaria chorismate synthase indicate their important role in protein structure and function

Predicted protein sequences of Chorismate synthase (Cs) from different Plasmodium species have sh... more Predicted protein sequences of Chorismate synthase (Cs) from different Plasmodium species have shown high number of conserved cysteine residues when compared to the predicted Cs protein sequences from other bacterial, fungal or plant species. To better understand the structure and function of malaria Cs, we have cloned, expressed and purified recombinant Cs from Plasmodium falciparum (rPfCs) in E.coli. the rPfCs exhibited disulfide linkages as indicated by mobility shifts observed for this protein when compared between non-reduced and reduced-alkylated conditions on SDS-PAGE. Antibodies generated against rPfCs also detected similar mobility shift for the native parasite protein from asexual blood stage culture indicating that the conserved cysteine residues in native parasite Cs play an important role in the protein structure and function.

Research paper thumbnail of Capsid-like particles decorated with the SARS2-CoV-2 receptor-binding domain elicit strong virus neutralization activity

The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we developed two capsid... more The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we developed two capsid-like particle (CLP)-based vaccines displaying the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. RBD antigens were displayed on AP205 CLPs through a novel split-protein Tag/Catcher ensuring unidirectional and high-density display of RBD. Both soluble recombinant RBD, and RBD displayed on CLPs bound the ACE2 receptor with nanomolar affinity. Mice were vaccinated with soluble RBD or CLP-displayed RBD, formulated in Squalene-Water-Emulsion. The RBD-CLP vaccines induced higher levels of serum anti-RBD antibodies, than the soluble RBD vaccines. Remarkably, one injection with our lead RBD-CLP vaccine in mice elicited virus neutralization antibody titers comparable to those found in patients which had recovered from Covid-19. Following booster vaccinations, the virus neutralization titers exceeded those measured after natural infection, at serum dilutions above 1:10.000. Thus, ...

Research paper thumbnail of Chorismate synthase from malaria parasites is bifunctional enzyme

Molecular and Biochemical Parasitology, 2019

Research paper thumbnail of A novel Pfs38 protein complex on the surface of Plasmodium falciparum blood-stage merozoites

Malaria journal, Feb 16, 2017

The Plasmodium genome encodes for a number of 6-Cys proteins that contain a module of six cystein... more The Plasmodium genome encodes for a number of 6-Cys proteins that contain a module of six cysteine residues forming three intramolecular disulphide bonds. These proteins have been well characterized at transmission as well as hepatic stages of the parasite life cycle. In the present study, a large complex of 6-Cys proteins: Pfs41, Pfs38 and Pfs12 and three other merozoite surface proteins: Glutamate-rich protein (GLURP), SERA5 and MSP-1 were identified on the Plasmodium falciparum merozoite surface. Recombinant 6-cys proteins i.e. Pfs38, Pfs12, Pfs41 as well as PfMSP-165 were expressed and purified using Escherichia coli expression system and antibodies were raised against each of these proteins. These antibodies were used to immunoprecipitate the native proteins and their associated partners from parasite lysate. ELISA, Far western, surface plasmon resonance and glycerol density gradient fractionation were carried out to confirm the respective interactions. Furthermore, erythrocyte...

Research paper thumbnail of Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition

Malaria Journal, 2012

Background Functional in vitro assays could provide insights into the efficacy of malaria vaccine... more Background Functional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays. Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos) was used for obtaining reliable live parasite counts through flow cytometry. Methods Both asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination of Giemsa-stained thin smears. A comparison of the ability of CMXRos to stain live and compromised para...

Research paper thumbnail of Optimisation and standardisation of a multiplex immunoassay of diverse Plasmodium falciparum antigens to assess changes in malaria transmission using sero-epidemiology

Wellcome Open Research, 2019

Background: Antibody responses have been used to characterise transmission and exposure history i... more Background: Antibody responses have been used to characterise transmission and exposure history in malaria-endemic settings for over a decade. Such studies have typically been conducted on well-standardised enzyme-linked immunosorbent assays (ELISAs). However, recently developed quantitative suspension array technologies (qSAT) are now capable of high-throughput and multiplexed screening of up to hundreds of analytes at a time. This study presents a customised protocol for the Luminex MAGPIX© qSAT using a diverse set of malaria antigens. The aim is to develop a standardised assay for routine serological surveillance that is implementable across laboratories and epidemiological settings. Methods: A panel of eight Plasmodium falciparum recombinant antigens, associated with long- and short-lived antibody responses, was designed for the Luminex MAGPIX© platform. The assay was optimised for key steps in the protocol: antigen-bead coupling concentration, buffer composition, serum sample d...

Research paper thumbnail of Preclinical development of a Pfs230-Pfs48/45 chimeric malaria transmission-blocking vaccine

npj Vaccines

The Plasmodium falciparum Pfs230 and Pfs48/45 proteins are leading candidates for a malaria trans... more The Plasmodium falciparum Pfs230 and Pfs48/45 proteins are leading candidates for a malaria transmission-blocking vaccine (TBV). Previously, we showed that a Pfs230–Pfs48/45 fusion protein elicits higher levels of functional antibodies than the individual antigens, but low yields hampered progression to clinical evaluation. Here we identified a modified construct (ProC6C) with a circumsporozoite protein (CSP) repeat-linker sequence that enhances expression. A scalable and reproducible process in the Lactococcus lactis expression system was developed and ProC6C was successfully transferred for manufacturing under current Good Manufacturing Practices (cGMP). In addition, a panel of analytical assays for release and stability were developed. Intact mass spectrometry analysis and multiangle light scattering showed that the protein contained correct disulfide bonds and was monomeric. Immunogenicity studies in mice showed that the ProC6C adsorbed to Alhydrogel®, with or without Matrix-MTM...

Research paper thumbnail of Capsid-like particles decorated with the SARS-CoV-2 receptor-binding domain elicit strong virus neutralization activity

Nature Communications

The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we develop two capsid-l... more The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we develop two capsid-like particle (CLP)-based vaccines displaying the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. RBD antigens are displayed on AP205 CLPs through a split-protein Tag/Catcher, ensuring unidirectional and high-density display of RBD. Both soluble recombinant RBD and RBD displayed on CLPs bind the ACE2 receptor with nanomolar affinity. Mice are vaccinated with soluble RBD or CLP-displayed RBD, formulated in Squalene-Water-Emulsion. The RBD-CLP vaccines induce higher levels of serum anti-spike antibodies than the soluble RBD vaccines. Remarkably, one injection with our lead RBD-CLP vaccine in mice elicits virus neutralization antibody titers comparable to those found in patients that had recovered from COVID-19. Following booster vaccinations, the virus neutralization titers exceed those measured after natural infection, at serum dilutions above 1:10,000. Thus, the RBD-CLP vac...

Research paper thumbnail of Suitability of IgG responses to multiple Plasmodium falciparum antigens as markers of transmission intensity and pattern

PLOS ONE

Detection of antibody reactivity to appropriate, specific parasite antigens may constitute a sens... more Detection of antibody reactivity to appropriate, specific parasite antigens may constitute a sensitive and cost-effective alternative to current tools to monitor malaria transmission across different endemicity settings. This study aimed to determine the suitability of IgG responses to a number of P. falciparum antigens as markers of transmission intensity and pattern. Antibody responses to multiple malaria antigens were determined in 905 participants aged 1–12 years from three districts with low (Keta), medium (Hohoe) and high (Krachi) transmission intensity in the Volta region of Ghana. Blood film microscopy slides and dry blood spots (DBS) were obtained for parasitaemia detection and antibody measurement, respectively. Sera were eluted from DBS and levels of IgG specific for 10 malaria antigens determined by a multiplex assay. Results were compared within and among the districts. Total IgG responses to MSPDBL1, MSPDBLLeucine, MSP2-FC27, RAMA, and PfRh2a and PfRh2b were higher in ...

Research paper thumbnail of A Reproducible and Scalable Process for Manufacturing a Pfs48/45 Based Plasmodium falciparum Transmission-Blocking Vaccine

Frontiers in Immunology

The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has bee... more The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has been advancing as a candidate antigen for a transmission-blocking vaccine (TBV) for malaria. However, Pfs48/45 contains multiple disulfide bonds, that are critical for proper folding and induction of transmission-blocking (TB) antibodies. We have previously shown that R0.6C, a fusion of the 6C domain of Pfs48/45 and a fragment of PfGLURP (R0), expressed in Lactococcus lactis, was properly folded and induced transmission-blocking antibodies. Here we describe the process development and technology transfer of a scalable and reproducible process suitable for R0.6C manufacturing under current Good Manufacturing Practices (cGMP). This process resulted in a final purified yield of 25 mg/L, sufficient for clinical evaluation. A panel of analytical assays for release and stability assessment of R0.6C were developed including HPLC, SDS-PAGE, and immunoblotting with the conformation-dependent TB mAb4...

Research paper thumbnail of Antibody Responses Against Plasmodium falciparum MSP3 Protein During Natural Malaria Infection in Individuals Living in Malaria-Endemic Regions of India

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences

Research paper thumbnail of GMZ2 Vaccine-Induced Antibody Responses, Naturally Acquired Immunity and the Incidence of Malaria in Burkinabe Children

Frontiers in Immunology

GMZ2 is a malaria vaccine candidate evaluated in a phase 2b multi-centre trial. Here we assessed ... more GMZ2 is a malaria vaccine candidate evaluated in a phase 2b multi-centre trial. Here we assessed antibody responses and the association of naturally acquired immunity with incidence of malaria in one of the trial sites, Banfora in Burkina Faso. The analysis included 453 (GMZ2 = 230, rabies = 223) children aged 12-60 months old. Children were followed-up for clinical malaria episodes for 12 months after final vaccine administration. Antibody levels against GMZ2 and eleven non-GMZ2 antigens were measured on days 0 and 84 (one month after final vaccine dose). Vaccine efficacy (VE) differed by age group (interaction, (12-35 months compared to 36-60 months), p = 0.0615). During the twelve months of follow-up, VE was 1% (95% confidence interval [CI] -17%, 17%) and 23% ([CI] 3%, 40%) in the 12 - 35 and 36 – 60 months old children, respectively. In the GMZ2 group, day 84 anti-GMZ2 IgG levels were associated with reduced incidence of febrile malaria during the follow up periods of 1-6 months...

Research paper thumbnail of Peripheral Merozoite Surface Proteins Are Targets of Naturally Acquired Immunity against Malaria in both India and Ghana

Infection and Immunity, 2020

Development of a successful blood-stage vaccine against Plasmodium falciparum malaria remains a h... more Development of a successful blood-stage vaccine against Plasmodium falciparum malaria remains a high priority. Immune-epidemiological studies are effective tools for the identification of antigenic targets of naturally acquired immunity (NAI) against malaria. However, differences in study design and methodology may compromise interstudy comparisons.

Research paper thumbnail of Natural antibody responses to Plasmodium falciparum MSP3 and GLURP(R0) antigens are associated with low parasite densities in malaria patients living in the Central Region of Ghana

Parasites & Vectors, 2017

Research paper thumbnail of Naturally Acquired Antibodies Target the Glutamate-Rich Protein on Intact Merozoites and Predict Protection Against Febrile Malaria

The Journal of Infectious Diseases, 2017

Research paper thumbnail of Naturally acquired antibody response to Plasmodium falciparum describes heterogeneity in transmission on islands in Lake Victoria

Scientific Reports, 2017

As markers of exposure anti-malaria antibody responses can help characterise heterogeneity in mal... more As markers of exposure anti-malaria antibody responses can help characterise heterogeneity in malaria transmission. In the present study antibody responses to Plasmodium falciparum AMA-1, MSP-119 and CSP were measured with the aim to describe transmission patterns in meso-endemic settings in Lake Victoria. Two cross-sectional surveys were conducted in Lake Victoria in January and August 2012. The study area comprised of three settings: mainland (Ungoye), large island (Mfangano) and small islands (Takawiri, Kibuogi, Ngodhe). Individuals provided a finger-blood sample to assess malaria infection by microscopy and PCR. Antibody response to P. falciparum was determined in 4,112 individuals by ELISA using eluted dried blood from filter paper. The overall seroprevalence was 64.0% for AMA-1, 39.5% for MSP-119, and 12.9% for CSP. Between settings, seroprevalences for merozoite antigens were similar between Ungoye and Mfangano, but higher when compared to the small islands. For AMA-1, the se...

Research paper thumbnail of Heterologous Expression and Evaluation of Novel Plasmodium falciparum Transmission Blocking Vaccine Candidates

Frontiers in Immunology

Malaria transmission blocking vaccines (TBV) aim to induce antibodies that can interrupt Plasmodi... more Malaria transmission blocking vaccines (TBV) aim to induce antibodies that can interrupt Plasmodium falciparum development in the mosquito midgut and thereby prevent onward malaria transmission. A limited number of TBV candidates have been identified and only three (Pfs25, Pfs230 and Pfs48/45) have entered clinical testing. While one of these candidates may emerge as a highly potent TBV candidate, it is premature to determine if they will generate sufficiently potent and sustained responses. It is therefore important to explore novel candidate antigens. We recently analyzed sera from naturally exposed individuals and found that the presence and/or intensity of antibodies against 12 novel putative surface expressed gametocyte antigens was associated with transmission reducing activity. In this study, protein fragments of these novel TBV candidates were designed and heterologously expressed in Drosophila melanogaster S2 cells and Lactococcus lactis. Eleven protein fragments, covering ...

Research paper thumbnail of Additional file 3 of Antibody responses to a suite of novel serological markers for malaria surveillance demonstrate strong correlation with clinical and parasitological infection across seasons and transmission settings in The Gambia

Additional file 3. Map of household geolocation of PfAMA1 sero-positive, clinical malaria and Pf ... more Additional file 3. Map of household geolocation of PfAMA1 sero-positive, clinical malaria and Pf infections across four villages during dry and wet transmission seasons. Spatial distribution of infections shown for lower transmission (N'demban) and higher transmission (Besse) villages in the West Coast Region (WCR) and lower transmission (Njaiyal) and higher transmission (Madina Samako) villages in the Upper River Region (URR). Infections at the start of the wet season (June – July 2013) shown on the left and during the wet and transmission season (August – December 2013) on the right.

Research paper thumbnail of Method for Production of Cysteine-Rich Proteins in Lactococcus lactis Expression System

Methods in Molecular Biology, 2022

The Gram-positive bacterium Lactococcus lactis is an ideal expression host for the overproduction... more The Gram-positive bacterium Lactococcus lactis is an ideal expression host for the overproduction of heterologous proteins in a functional form. L. lactis has recently been identified as an efficient Gram-positive cell factory for the production of recombinant proteins and the safety of this production system has been confirmed in multiple clinical trials. Key desirable features of L. lactis include its generally recognized as safe (GRAS) status, long history of safe use in food production, probiotic properties, absence of endotoxins, capacity to secrete stable recombinant protein to the growth medium, the presence of few proteases, and a diverse selection of cloning and inducible expression vectors. Growth of lactococci is rapid, proceeds to high cell densities, and does not require aeration, which facilitates large-scale fermentation. We have previously described the production of several Plasmodium falciparum antigens with varying degrees of predicted structural complexities, those which are considered difficult-to-produce proteins by using L. lactis pH-dependent inducible promoter (P170). The purpose of this chapter is to provide a detailed protocol for the expression of difficult-to-produce proteins, mainly high cysteine-rich proteins, in the soluble form in L. lactis from cloning of the target gene to the determination of expression levels and purification.

Research paper thumbnail of Data from: Unravelling the immune signature of Plasmodium falciparum transmission-reducing immunity

Infection with Plasmodium can elicit antibodies that inhibit parasite survival in the mosquito, w... more Infection with Plasmodium can elicit antibodies that inhibit parasite survival in the mosquito, when they are ingested in an infectious blood meal. Here, we determine the transmission-reducing activity (TRA) of naturally acquired antibodies from 648 malaria-exposed individuals using lab-based mosquito-feeding assays. Transmission inhibition is significantly associated with antibody responses to Pfs48/45, Pfs230, and to 43 novel gametocyte proteins assessed by protein microarray. In field-based mosquito-feeding assays the likelihood and rate of mosquito infection are significantly lower for individuals reactive to Pfs48/45, Pfs230 or to combinations of the novel TRA-associated proteins. We also show that naturally acquired purified antibodies against key transmission-blocking epitopes of Pfs48/45 and Pfs230 are mechanistically involved in TRA, whereas sera depleted of these antibodies retain high-level, complement-independent TRA. Our analysis demonstrates that host antibody response...

Research paper thumbnail of Conserved cysteine residues in malaria chorismate synthase indicate their important role in protein structure and function

Predicted protein sequences of Chorismate synthase (Cs) from different Plasmodium species have sh... more Predicted protein sequences of Chorismate synthase (Cs) from different Plasmodium species have shown high number of conserved cysteine residues when compared to the predicted Cs protein sequences from other bacterial, fungal or plant species. To better understand the structure and function of malaria Cs, we have cloned, expressed and purified recombinant Cs from Plasmodium falciparum (rPfCs) in E.coli. the rPfCs exhibited disulfide linkages as indicated by mobility shifts observed for this protein when compared between non-reduced and reduced-alkylated conditions on SDS-PAGE. Antibodies generated against rPfCs also detected similar mobility shift for the native parasite protein from asexual blood stage culture indicating that the conserved cysteine residues in native parasite Cs play an important role in the protein structure and function.

Research paper thumbnail of Capsid-like particles decorated with the SARS2-CoV-2 receptor-binding domain elicit strong virus neutralization activity

The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we developed two capsid... more The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we developed two capsid-like particle (CLP)-based vaccines displaying the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. RBD antigens were displayed on AP205 CLPs through a novel split-protein Tag/Catcher ensuring unidirectional and high-density display of RBD. Both soluble recombinant RBD, and RBD displayed on CLPs bound the ACE2 receptor with nanomolar affinity. Mice were vaccinated with soluble RBD or CLP-displayed RBD, formulated in Squalene-Water-Emulsion. The RBD-CLP vaccines induced higher levels of serum anti-RBD antibodies, than the soluble RBD vaccines. Remarkably, one injection with our lead RBD-CLP vaccine in mice elicited virus neutralization antibody titers comparable to those found in patients which had recovered from Covid-19. Following booster vaccinations, the virus neutralization titers exceeded those measured after natural infection, at serum dilutions above 1:10.000. Thus, ...

Research paper thumbnail of Chorismate synthase from malaria parasites is bifunctional enzyme

Molecular and Biochemical Parasitology, 2019

Research paper thumbnail of A novel Pfs38 protein complex on the surface of Plasmodium falciparum blood-stage merozoites

Malaria journal, Feb 16, 2017

The Plasmodium genome encodes for a number of 6-Cys proteins that contain a module of six cystein... more The Plasmodium genome encodes for a number of 6-Cys proteins that contain a module of six cysteine residues forming three intramolecular disulphide bonds. These proteins have been well characterized at transmission as well as hepatic stages of the parasite life cycle. In the present study, a large complex of 6-Cys proteins: Pfs41, Pfs38 and Pfs12 and three other merozoite surface proteins: Glutamate-rich protein (GLURP), SERA5 and MSP-1 were identified on the Plasmodium falciparum merozoite surface. Recombinant 6-cys proteins i.e. Pfs38, Pfs12, Pfs41 as well as PfMSP-165 were expressed and purified using Escherichia coli expression system and antibodies were raised against each of these proteins. These antibodies were used to immunoprecipitate the native proteins and their associated partners from parasite lysate. ELISA, Far western, surface plasmon resonance and glycerol density gradient fractionation were carried out to confirm the respective interactions. Furthermore, erythrocyte...

Research paper thumbnail of Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition

Malaria Journal, 2012

Background Functional in vitro assays could provide insights into the efficacy of malaria vaccine... more Background Functional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays. Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos) was used for obtaining reliable live parasite counts through flow cytometry. Methods Both asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination of Giemsa-stained thin smears. A comparison of the ability of CMXRos to stain live and compromised para...

Research paper thumbnail of Optimisation and standardisation of a multiplex immunoassay of diverse Plasmodium falciparum antigens to assess changes in malaria transmission using sero-epidemiology

Wellcome Open Research, 2019

Background: Antibody responses have been used to characterise transmission and exposure history i... more Background: Antibody responses have been used to characterise transmission and exposure history in malaria-endemic settings for over a decade. Such studies have typically been conducted on well-standardised enzyme-linked immunosorbent assays (ELISAs). However, recently developed quantitative suspension array technologies (qSAT) are now capable of high-throughput and multiplexed screening of up to hundreds of analytes at a time. This study presents a customised protocol for the Luminex MAGPIX© qSAT using a diverse set of malaria antigens. The aim is to develop a standardised assay for routine serological surveillance that is implementable across laboratories and epidemiological settings. Methods: A panel of eight Plasmodium falciparum recombinant antigens, associated with long- and short-lived antibody responses, was designed for the Luminex MAGPIX© platform. The assay was optimised for key steps in the protocol: antigen-bead coupling concentration, buffer composition, serum sample d...

Research paper thumbnail of Preclinical development of a Pfs230-Pfs48/45 chimeric malaria transmission-blocking vaccine

npj Vaccines

The Plasmodium falciparum Pfs230 and Pfs48/45 proteins are leading candidates for a malaria trans... more The Plasmodium falciparum Pfs230 and Pfs48/45 proteins are leading candidates for a malaria transmission-blocking vaccine (TBV). Previously, we showed that a Pfs230–Pfs48/45 fusion protein elicits higher levels of functional antibodies than the individual antigens, but low yields hampered progression to clinical evaluation. Here we identified a modified construct (ProC6C) with a circumsporozoite protein (CSP) repeat-linker sequence that enhances expression. A scalable and reproducible process in the Lactococcus lactis expression system was developed and ProC6C was successfully transferred for manufacturing under current Good Manufacturing Practices (cGMP). In addition, a panel of analytical assays for release and stability were developed. Intact mass spectrometry analysis and multiangle light scattering showed that the protein contained correct disulfide bonds and was monomeric. Immunogenicity studies in mice showed that the ProC6C adsorbed to Alhydrogel®, with or without Matrix-MTM...

Research paper thumbnail of Capsid-like particles decorated with the SARS-CoV-2 receptor-binding domain elicit strong virus neutralization activity

Nature Communications

The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we develop two capsid-l... more The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we develop two capsid-like particle (CLP)-based vaccines displaying the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. RBD antigens are displayed on AP205 CLPs through a split-protein Tag/Catcher, ensuring unidirectional and high-density display of RBD. Both soluble recombinant RBD and RBD displayed on CLPs bind the ACE2 receptor with nanomolar affinity. Mice are vaccinated with soluble RBD or CLP-displayed RBD, formulated in Squalene-Water-Emulsion. The RBD-CLP vaccines induce higher levels of serum anti-spike antibodies than the soluble RBD vaccines. Remarkably, one injection with our lead RBD-CLP vaccine in mice elicits virus neutralization antibody titers comparable to those found in patients that had recovered from COVID-19. Following booster vaccinations, the virus neutralization titers exceed those measured after natural infection, at serum dilutions above 1:10,000. Thus, the RBD-CLP vac...

Research paper thumbnail of Suitability of IgG responses to multiple Plasmodium falciparum antigens as markers of transmission intensity and pattern

PLOS ONE

Detection of antibody reactivity to appropriate, specific parasite antigens may constitute a sens... more Detection of antibody reactivity to appropriate, specific parasite antigens may constitute a sensitive and cost-effective alternative to current tools to monitor malaria transmission across different endemicity settings. This study aimed to determine the suitability of IgG responses to a number of P. falciparum antigens as markers of transmission intensity and pattern. Antibody responses to multiple malaria antigens were determined in 905 participants aged 1–12 years from three districts with low (Keta), medium (Hohoe) and high (Krachi) transmission intensity in the Volta region of Ghana. Blood film microscopy slides and dry blood spots (DBS) were obtained for parasitaemia detection and antibody measurement, respectively. Sera were eluted from DBS and levels of IgG specific for 10 malaria antigens determined by a multiplex assay. Results were compared within and among the districts. Total IgG responses to MSPDBL1, MSPDBLLeucine, MSP2-FC27, RAMA, and PfRh2a and PfRh2b were higher in ...

Research paper thumbnail of A Reproducible and Scalable Process for Manufacturing a Pfs48/45 Based Plasmodium falciparum Transmission-Blocking Vaccine

Frontiers in Immunology

The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has bee... more The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has been advancing as a candidate antigen for a transmission-blocking vaccine (TBV) for malaria. However, Pfs48/45 contains multiple disulfide bonds, that are critical for proper folding and induction of transmission-blocking (TB) antibodies. We have previously shown that R0.6C, a fusion of the 6C domain of Pfs48/45 and a fragment of PfGLURP (R0), expressed in Lactococcus lactis, was properly folded and induced transmission-blocking antibodies. Here we describe the process development and technology transfer of a scalable and reproducible process suitable for R0.6C manufacturing under current Good Manufacturing Practices (cGMP). This process resulted in a final purified yield of 25 mg/L, sufficient for clinical evaluation. A panel of analytical assays for release and stability assessment of R0.6C were developed including HPLC, SDS-PAGE, and immunoblotting with the conformation-dependent TB mAb4...