Publications by authors named "Ankit Dwivedi"

10 Publications

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Strains used in whole organism Plasmodium falciparum vaccine trials differ in genome structure, sequence, and immunogenic potential.

Genome Med 2020 01 8;12(1). Epub 2020 Jan 8.

Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.

Background: Plasmodium falciparum (Pf) whole-organism sporozoite vaccines have been shown to provide significant protection against controlled human malaria infection (CHMI) in clinical trials. Initial CHMI studies showed significantly higher durable protection against homologous than heterologous strains, suggesting the presence of strain-specific vaccine-induced protection. However, interpretation of these results and understanding of their relevance to vaccine efficacy have been hampered by the lack of knowledge on genetic differences between vaccine and CHMI strains, and how these strains are related to parasites in malaria endemic regions.

Methods: Whole genome sequencing using long-read (Pacific Biosciences) and short-read (Illumina) sequencing platforms was conducted to generate de novo genome assemblies for the vaccine strain, NF54, and for strains used in heterologous CHMI (7G8 from Brazil, NF166.C8 from Guinea, and NF135.C10 from Cambodia). The assemblies were used to characterize sequences in each strain relative to the reference 3D7 (a clone of NF54) genome. Strains were compared to each other and to a collection of clinical isolates (sequenced as part of this study or from public repositories) from South America, sub-Saharan Africa, and Southeast Asia.

Results: While few variants were detected between 3D7 and NF54, we identified tens of thousands of variants between NF54 and the three heterologous strains. These variants include SNPs, indels, and small structural variants that fall in regulatory and immunologically important regions, including transcription factors (such as PfAP2-L and PfAP2-G) and pre-erythrocytic antigens that may be key for sporozoite vaccine-induced protection. Additionally, these variants directly contributed to diversity in immunologically important regions of the genomes as detected through in silico CD8 T cell epitope predictions. Of all heterologous strains, NF135.C10 had the highest number of unique predicted epitope sequences when compared to NF54. Comparison to global clinical isolates revealed that these four strains are representative of their geographic origin despite long-term culture adaptation; of note, NF135.C10 is from an admixed population, and not part of recently formed subpopulations resistant to artemisinin-based therapies present in the Greater Mekong Sub-region.

Conclusions: These results will assist in the interpretation of vaccine efficacy of whole-organism vaccines against homologous and heterologous CHMI.
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January 2020

A sporozoite-based vaccination platform against human malaria.

NPJ Vaccines 2018 24;3:33. Epub 2018 Aug 24.

1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal.

There is a pressing need for safe and highly effective () malaria vaccines. The circumsporozoite protein (CS), expressed on sporozoites and during early hepatic stages, is a leading target vaccine candidate, but clinical efficacy has been modest so far. Conversely, whole-sporozoite (WSp) vaccines have consistently shown high levels of sterilizing immunity and constitute a promising approach to effective immunization against malaria. Here, we describe a novel WSp malaria vaccine that employs transgenic sporozoites of rodent () parasites as cross-species immunizing agents and as platforms for expression and delivery of CS (Vac). We show that both wild-type and Vac sporozoites unabatedly infect and develop in human hepatocytes while unable to establish an infection in human red blood cells. In a rabbit model, similarly susceptible to hepatic but not blood infection, we show that Vac elicits cross-species cellular immune responses, as well as CS-specific antibodies that efficiently inhibit sporozoite liver invasion in human hepatocytes and in mice with humanized livers. Thus, Vac is safe and induces functional immune responses in preclinical studies, warranting clinical testing and development.
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August 2018

Functional analysis of Plasmodium falciparum subpopulations associated with artemisinin resistance in Cambodia.

Malar J 2017 Dec 19;16(1):493. Epub 2017 Dec 19.

Institut de Biologie Computationnelle (IBC), 34095, Montpellier, France.

Background: Plasmodium falciparum malaria is one of the most widespread parasitic infections in humans and remains a leading global health concern. Malaria elimination efforts are threatened by the emergence and spread of resistance to artemisinin-based combination therapy, the first-line treatment of malaria. Promising molecular markers and pathways associated with artemisinin drug resistance have been identified, but the underlying molecular mechanisms of resistance remains unknown. The genomic data from early period of emergence of artemisinin resistance (2008-2011) was evaluated, with aim to define k13 associated genetic background in Cambodia, the country identified as epicentre of anti-malarial drug resistance, through characterization of 167 parasite isolates using a panel of 21,257 SNPs.

Results: Eight subpopulations were identified suggesting a process of acquisition of artemisinin resistance consistent with an emergence-selection-diffusion model, supported by the shifting balance theory. Identification of population specific mutations facilitated the characterization of a core set of 57 background genes associated with artemisinin resistance and associated pathways. The analysis indicates that the background of artemisinin resistance was not acquired after drug pressure, rather is the result of fixation followed by selection on the daughter subpopulations derived from the ancestral population.

Conclusions: Functional analysis of artemisinin resistance subpopulations illustrates the strong interplay between ubiquitination and cell division or differentiation in artemisinin resistant parasites. The relationship of these pathways with the P. falciparum resistant subpopulation and presence of drug resistance markers in addition to k13, highlights the major role of admixed parasite population in the diffusion of artemisinin resistant background. The diffusion of resistant genes in the Cambodian admixed population after selection resulted from mating of gametocytes of sensitive and resistant parasite populations.
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December 2017

Association of a Novel Mutation in the Plasmodium falciparum Chloroquine Resistance Transporter With Decreased Piperaquine Sensitivity.

J Infect Dis 2017 08;216(4):468-476

Division of Malaria Research, Institute for Global Health.

Background: Amplified copy number in the plasmepsin II/III genes within Plasmodium falciparum has been associated with decreased sensitivity to piperaquine. To examine this association and test whether additional loci might also contribute, we performed a genome-wide association study of ex vivo P. falciparum susceptibility to piperaquine.

Methods: Plasmodium falciparum DNA from 183 samples collected primarily from Cambodia was genotyped at 33716 genome-wide single nucleotide polymorphisms (SNPs). Linear mixed models and random forests were used to estimate associations between parasite genotypes and piperaquine susceptibility. Candidate polymorphisms were evaluated for their association with dihydroartemisinin-piperaquine treatment outcomes in an independent dataset.

Results: Single nucleotide polymorphisms on multiple chromosomes were associated with piperaquine 90% inhibitory concentrations (IC90) in a genome-wide analysis. Fine-mapping of genomic regions implicated in genome-wide analyses identified multiple SNPs in linkage disequilibrium with each other that were significantly associated with piperaquine IC90, including a novel mutation within the gene encoding the P. falciparum chloroquine resistance transporter, PfCRT. This mutation (F145I) was associated with dihydroartemisinin-piperaquine treatment failure after adjusting for the presence of amplified plasmepsin II/III, which was also associated with decreased piperaquine sensitivity.

Conclusions: Our data suggest that, in addition to plasmepsin II/III copy number, other loci, including pfcrt, may also be involved in piperaquine resistance.
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August 2017

Valine/isoleucine variants drive selective pressure in the VP1 sequence of EV-A71 enteroviruses.

BMC Infect Dis 2017 05 8;17(1):333. Epub 2017 May 8.

Cirad, UMR 17, Intertryp, TA-A17/G, Campus International de Baillarguet, 34398, Montpellier Cedex 5, France.

Background: In 2011-2012, Northern Vietnam experienced its first large scale hand foot and mouth disease (HFMD) epidemic. In 2011, a major HFMD epidemic was also reported in South Vietnam with fatal cases. This 2011-2012 outbreak was the first one to occur in North Vietnam providing grounds to study the etiology, origin and dynamic of the disease. We report here the analysis of the VP1 gene of strains isolated throughout North Vietnam during the 2011-2012 outbreak and before.

Methods: The VP1 gene of 106 EV-A71 isolates from North Vietnam and 2 from Central Vietnam were sequenced. Sequence alignments were analyzed at the nucleic acid and protein level. Gene polymorphism was also analyzed. A Factorial Correspondence Analysis was performed to correlate amino acid mutations with clinical parameters.

Results: The sequences were distributed into four phylogenetic clusters. Three clusters corresponded to the subgenogroup C4 and the last one corresponded to the subgenogroup C5. Each cluster displayed different polymorphism characteristics. Proteins were highly conserved but three sites bearing only Isoleucine (I) or Valine (V) were characterized. The isoleucine/valine variability matched the clusters. Spatiotemporal analysis of the I/V variants showed that all variants which emerged in 2011 and then in 2012 were not the same but were all present in the region prior to the 2011-2012 outbreak. Some correlation was found between certain I/V variants and ethnicity and severity.

Conclusions: The 2011-2012 outbreak was not caused by an exogenous strain coming from South Vietnam or elsewhere but by strains already present and circulating at low level in North Vietnam. However, what triggered the outbreak remains unclear. A selective pressure is applied on I/V variants which matches the genetic clusters. I/V variants were shown on other viruses to correlate with pathogenicity. This should be investigated in EV-A71. I/V variants are an easy and efficient way to survey and identify circulating EV-A71 strains.
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May 2017

Babesia microti from humans and ticks hold a genomic signature of strong population structure in the United States.

BMC Genomics 2016 11 7;17(1):888. Epub 2016 Nov 7.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, 06520, USA.

Background: Babesia microti is an emerging tick-borne apicomplexan parasite with increasing geographic range and incidence in the United States. The rapid expansion of B. microti into its current distribution in the northeastern USA has been due to the range expansion of the tick vector, Ixodes scapularis, upon which the causative agent is dependent for transmission to humans.

Results: To reconstruct the history of B. microti in the continental USA and clarify the evolutionary origin of human strains, we used multiplexed hybrid capture of 25 B. microti isolates obtained from I. scapularis and human blood. Despite low genomic variation compared with other Apicomplexa, B. microti was strongly structured into three highly differentiated genetic clusters in the northeastern USA. Bayesian analyses of the apicoplast genomes suggest that the origin of the current diversity of B. microti in northeastern USA dates back 46 thousand years with a signature of recent population expansion in the last 1000 years. Human-derived samples belonged to two rarely intermixing clusters, raising the possibility of highly divergent infectious phenotypes in humans.

Conclusions: Our results validate the multiplexed hybrid capture strategy for characterizing genome-wide diversity and relatedness of B. microti from ticks and humans. We find strong population structure in B. microti samples from the Northeast indicating potential barriers to gene flow.
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November 2016

Genome-wide diversity and gene expression profiling of Babesia microti isolates identify polymorphic genes that mediate host-pathogen interactions.

Sci Rep 2016 10 18;6:35284. Epub 2016 Oct 18.

Antigen Discovery Inc., Irvine, CA, 92618 USA.

Babesia microti, a tick-transmitted, intraerythrocytic protozoan parasite circulating mainly among small mammals, is the primary cause of human babesiosis. While most cases are transmitted by Ixodes ticks, the disease may also be transmitted through blood transfusion and perinatally. A comprehensive analysis of genome composition, genetic diversity, and gene expression profiling of seven B. microti isolates revealed that genetic variation in isolates from the Northeast United States is almost exclusively associated with genes encoding the surface proteome and secretome of the parasite. Furthermore, we found that polymorphism is restricted to a small number of genes, which are highly expressed during infection. In order to identify pathogen-encoded factors involved in host-parasite interactions, we screened a proteome array comprised of 174 B. microti proteins, including several predicted members of the parasite secretome. Using this immuno-proteomic approach we identified several novel antigens that trigger strong host immune responses during the onset of infection. The genomic and immunological data presented herein provide the first insights into the determinants of B. microti interaction with its mammalian hosts and their relevance for understanding the selective pressures acting on parasite evolution.
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October 2016

Plasmodium falciparum parasite population structure and gene flow associated to anti-malarial drugs resistance in Cambodia.

Malar J 2016 06 14;15:319. Epub 2016 Jun 14.

Institut de Biologie Computationnelle (IBC), Montpellier, France.

Background: Western Cambodia is recognized as the epicentre of emergence of Plasmodium falciparum multi-drug resistance. The emergence of artemisinin resistance has been observed in this area since 2008-2009 and molecular signatures associated to artemisinin resistance have been characterized in k13 gene. At present, one of the major threats faced, is the possible spread of Asian artemisinin resistant parasites over the world threatening millions of people and jeopardizing malaria elimination programme efforts. To anticipate the diffusion of artemisinin resistance, the identification of the P. falciparum population structure and the gene flow among the parasite population in Cambodia are essential.

Methods: To this end, a mid-throughput PCR-LDR-FMA approach based on LUMINEX technology was developed to screen for genetic barcode in 533 blood samples collected in 2010-2011 from 16 health centres in malaria endemics areas in Cambodia.

Results: Based on successful typing of 282 samples, subpopulations were characterized along the borders of the country. Each 11-loci barcode provides evidence supporting allele distribution gradient related to subpopulations and gene flow. The 11-loci barcode successfully identifies recently emerging parasite subpopulations in western Cambodia that are associated with the C580Y dominant allele for artemisinin resistance in k13 gene. A subpopulation was identified in northern Cambodia that was associated to artemisinin (R539T resistant allele of k13 gene) and mefloquine resistance.

Conclusions: The gene flow between these subpopulations might have driven the spread of artemisinin resistance over Cambodia.
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June 2016

Breast cancer screening existence in India: A nonexisting reality.

Indian J Med Paediatr Oncol 2015 Oct-Dec;36(4):207-9

GPgy1 Internal Medicine Resident, St Barnabas Hospital, Bronx, New York, USA. E-mail:

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January 2016

Sequence and annotation of the apicoplast genome of the human pathogen Babesia microti.

PLoS One 2014 3;9(10):e107939. Epub 2014 Oct 3.

Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, Connecticut, United States of America.

The apicomplexan intraerythrocytic parasite Babesia microti is an emerging human pathogen and the primary cause of human babesiosis, a malaria-like illness endemic in the United States. The pathogen is transmitted to humans by the tick vector, Ixodes scapularis, and by transfusion of blood from asymptomatic B. microti-infected donors. Whereas the nuclear and mitochondrial genomes of this parasite have been sequenced, assembled and annotated, its apicoplast genome remained incomplete, mainly due to its low representation and high A+T content. Here we report the complete sequence and annotation of the apicoplast genome of the B. microti R1 isolate. The genome consists of a 28.7 kb circular molecule encoding primarily functions important for maintenance of the apicoplast DNA, transcription, translation and maturation of organellar proteins. Genome analysis and annotation revealed a unique gene structure and organization of the B. microti apicoplast genome and suggest that all metabolic and non-housekeeping functions in this organelle are nuclear-encoded. B. microti apicoplast functions are significantly different from those of the host, suggesting that they might be useful as targets for development of potent and safe therapies for the treatment of human babesiosis.
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July 2015