Biomedical subjects

Priti Kumar

Publications and source records attributed to Priti Kumar.

9 recordsLinked to original sources

PubMed2026

Development of a Recombinant Adeno-Associated Virus Vector for Human T Lymphocyte- and Natural Killer Cell-Targeted Gene Therapy.

Recombinant adeno-associated virus (rAAV) vectors are widely used for gene delivery but show limited efficiency in immune cells, including T lymphocytes and natural killer (NK) cells. To overcome this barrier, we have developed a CD7-targeted rAAV vector (CD7-AAV6/9) featuring a nanobody-fused hybrid capsid derived from a rationally selected chimeric combination of AAV6 and AAV9. CD7-AAV6/9 enables efficient and selective transduction of immortalized and primary human T and NK cells in vitro and in vivo in a humanized mouse model, achieves high production titers, and exhibits markedly reduced off-target transduction compared with wild-type serotypes. Incorporation of a human gene-derived intron into the vector genome to overcome host-mediated transcriptional repression enables robust transgene expression in human CD7+ T lymphocyte and NK cell populations. Together, our findings establish an integrated capsid-genome design framework for targeting human T and NK cells, notoriously challenging immune cell populations for gene therapy, and provide a versatile platform readily adaptable to alternative surface markers and therapeutic payloads.

NK cells↗

PubMed2006

Interfering antiviral immunity: application, subversion, hope?

RNA interference (RNAi), initially recognized as a natural antiviral mechanism in plants, has rapidly emerged as an invaluable tool to suppress gene expression in a sequence-specific manner in all organisms, including mammals. Its potential to inhibit the replication of a variety of viruses has been demonstrated in vitro and in vivo in mouse and monkey models. These results have generated profound interest in the use of this technology as a potential treatment strategy for viral infections for which vaccines and drugs are unavailable or inadequate. In this review, we discuss the progress made within the past 2-3 years towards harnessing the potential of RNAi for clinical application in viral infections and the hurdles that have yet to be overcome.

Animals↗

PubMed2006

A single siRNA suppresses fatal encephalitis induced by two different flaviviruses.

BACKGROUND: Japanese encephalitis virus (JEV) and West Nile virus (WNV) are neurotropic flaviviruses that can cause acute encephalitis with a high fatality rate. Currently there is no effective treatment for these infections. METHODS AND FINDINGS: We tested RNA interference (RNAi)-based intervention to suppress lethal JE and WN encephalitis in mice. To induce RNAi, we used either lentivirally expressed short hairpin RNA (shRNA) or synthetic short interfering RNA (siRNA). As target, we selected the cd loop-coding sequence in domain II of the viral Envelope protein, which is highly conserved among all flaviviruses because of its essential role in membrane fusion. Using as a target a species-specific sequence in the cd loop that is conserved only among the different strains of either JEV or WNV, we could achieve specific protection against the corresponding virus. However, by targeting a cross-species conserved sequence within the cd loop, we were able to protect mice against encephalitis induced by both viruses. A single intracranial administration of lentivirally delivered shRNA or lipid-complexed siRNA before viral challenge or siRNA treatment after viral challenge was sufficient for protection against lethal encephalitis. CONCLUSIONS: RNAi-based intervention affords near complete protection from both JEV- and WNV- induced encephalitis in mice. Our results show, to our knowledge for the first time, that siRNA can be used as a broad-spectrum antiviral agent for treating encephalitis caused by multiple related viruses.

Animals↗

PubMed2005

Lentiviral delivery of short hairpin RNAs protects CD4 T cells from multiple clades and primary isolates of HIV.

Viral heterogeneity is a major hurdle for potential therapeutic use of RNA interference (RNAi) against HIV-1. To determine the extent of RNAi tolerance to mutations, we tested 3 viral target sites with differing propensity for mutations: a highly variable rev sequence, a gag sequence conserved only among clade B isolates, and a vif sequence highly conserved across clades. Lentiviral expression of all 3 shRNAs inhibited replication of the homologous HIV(IIIB) strain. However, they differed in their ability to protect primary CD4 T cells against multiple isolates within and across HIV clades. The least conserved rev sequence inhibited only 2 of 5 clade B isolates. The gag sequence (conserved within clade B) protected 5 of 5 clade B isolates but not other clade viruses with 2 or 3 mutations in the central region. In contrast, the vif sequence, which was conserved across clades except for single mutations at positions 14 and 17, inhibited viruses from 5 different clades. Moreover, siRNAs with introduced mutations at sites of gag sequence polymorphisms showed reduced antiviral activity, whereas mutations in vif siRNA only modestly decreased silencing. Thus, although 1 or 2 mutations at peripheral sites are tolerated, mutations in the central target cleavage region abolish RNAi activity.

Antiviral Agents↗

PubMed2004

Impaired T helper 1 function of nonstructural protein 3-specific T cells in Japanese patients with encephalitis with neurological sequelae.

Japanese encephalitis virus (JEV) is the principal cause of viral encephalitis. The predominance of children among patients with encephalitis in areas where JEV is endemic that do not have immunization programs suggests that acquired immunity is critical in protecting adults against symptomatic JEV encephalitis. We characterized and compared the T cell response to nonstructural (NS) protein 3 between healthy individuals naturally exposed to JEV and patients in the convalescent phase of JEV encephalitis. The NS3 protein, used as a fusion to the 11-amino acid protein transduction domain of human immunodeficiency virus Tat, elicited CD4(+) and T helper-dependent CD8(+) T cell responses. The production of interferon (IFN)- gamma by responding T cells was significantly higher in healthy donors than in patients, correlating strongly with acquired protective immunity to JEV. Furthermore, a striking inverse association between IFN- gamma levels and the severity of postencephalitic sequelae in patients implicated a role of IFN- gamma in recovery.

Antibody Formation↗

PubMed2004

Cell-mediated immune responses in healthy children with a history of subclinical infection with Japanese encephalitis virus: analysis of CD4+ and CD8+ T cell target specificities by intracellular delivery of viral proteins using the human immunodeficiency virus Tat protein transduction domain.

Japanese encephalitis virus (JEV), a single-stranded positive-sense RNA virus of the family Flaviviridae, is the major cause of paediatric encephalitis in Asia. The high incidence of subclinical infections in Japanese encephalitis-endemic areas and subsequent evasion of encephalitis points to the development of immune responses against JEV. Humoral responses play a central role in protection against JEV; however, cell-mediated immune responses contributing to this end are not fully understood. The structural envelope (E) protein, the major inducer of neutralizing antibodies, is a poor target for T cells in natural JEV infections. The extent to which JEV non-structural proteins are targeted by T cells in subclinically infected healthy children would help to elucidate the role of cell-mediated immunity in protection against JEV as well as other flaviviral infections. The property of the Tat peptide of Human immunodeficiency virus to transduce proteins across cell membranes, facilitating intracellular protein delivery following exogenous addition to cultured cells, prompted us to express the four largest proteins of JEV, comprising 71 % of the JEV genome coding sequence, as Tat fusions for enumerating the frequencies of virus-specific CD4(+) and CD8(+) T cells in JEV-immune donors. At least two epitopes recognized by distinct HLA alleles were found on each of the non-structural proteins, with dominant antiviral Th1 T cell responses to the NS3 protein in nearly 96 % of the cohort. The data presented here show that non-structural proteins are frequently targeted by T cells in natural JEV infections and may be efficacious supplements for the predominantly antibody-eliciting E-based JEV vaccines.

Alleles↗

PubMed2004

Conserved amino acids 193-324 of non-structural protein 3 are a dominant source of peptide determinants for CD4+ and CD8+ T cells in a healthy Japanese encephalitis virus-endemic cohort.

Our earlier identification of the non-structural protein 3 (NS3) of Japanese encephalitis virus (JEV) as a dominant CD4+ as well as CD8+ T cell-eliciting antigen in a healthy JEV-endemic cohort with a wide HLA distribution implied the presence of several epitopes dispersed over the length of the protein. Use of various truncated versions of NS3 in lymphocyte stimulation and interferon (IFN)-gamma secretion assays revealed that amino acids (aa) 193-324 of NS3 were comparable with, if not superior to, the full-length protein in evoking Th1 responses. The potential of this 14.4 kDa stretch to stimulate IFN-gamma production from both subtypes of T cells in a manner qualitatively and quantitatively similar to the 68 kDa parent protein suggested the presence within it of both class I and II epitopes and demonstrated that the entire immunogenicity of NS3 was focused on aa 193-324. Interestingly, this segment contained five of the eight helicase motifs of NS3. Analysis of variability of the NS3 protein sequence across 16 JEV isolates revealed complete identity of aa 219-318, which is contained within the above segment, suggesting that NS3-specific epitopes tend to cluster in relatively conserved regions that harbour functionally critical domains of the protein.

Adolescent↗

PubMed2003

Health impact assessment needs in south-east Asian countries.

A situation analysis was undertaken to assess impediments to health impact assessment (HIA) in the South-East Asia Region of WHO (SEARO). The countries of the region were assessed on the policy framework and procedures for HIA, existing infrastructure required to support HIA, the capacity for undertaking HIA, and the potential for intersectoral collaboration. The findings show that environmental impact assessment (EIA) is being used implicitly as a substitute for HIA, which is not explicitly or routinely conducted in virtually all countries of the Region. Therefore, policy, infrastructure, capacity, and intersectoral collaboration need strengthening for the routine implementation of HIA.

Asia, Southeastern↗

PubMed2003

The Apa protein of Mycobacterium tuberculosis stimulates gamma interferon-secreting CD4+ and CD8+ T cells from purified protein derivative-positive individuals and affords protection in a guinea pig model.

The search to identify Mycobacterium tuberculosis antigens capable of conferring protective immunity against tuberculosis has received a boost owing to the resurgence of tuberculosis over the past two decades. It has long been recognized that lymphoid cells are required for protection against M. tuberculosis. While traditionally the CD4(+) populations of T cells were believed to predominantly serve this protective function, a pivotal role for CD8(+) T cells in this task has been increasingly appreciated. We show that the 50- to 55-kDa Apa protein, specified by the Rv1860 gene of M. tuberculosis, can elicit both lymphoproliferative response and gamma interferon (IFN-gamma) production from peripheral blood mononuclear cells (PBMC) of purified protein derivative (PPD)-positive individuals, with significant differences recorded in the levels of responsiveness between PPD-positive healthy controls and pulmonary tuberculosis patients. Flow cytometric analysis of whole blood stimulated with the recombinant Apa protein revealed a sizeable proportion of CD8(+) T cells in addition to CD4(+) T cells contributing to IFN-gamma secretion. PBMC responding to the Apa protein produced no interleukin-4, revealing a Th1 phenotype. A DNA vaccine and a poxvirus recombinant expressing the Apa protein were constructed and tested for their ability to protect immunized guinea pigs against a challenge dose of virulent M. tuberculosis. Although the DNA vaccine afforded little protection, the poxvirus recombinant boost after DNA vaccine priming conferred a significant level of protective immunity, bringing about a considerable reduction in mycobacterial counts from the challenge bacilli in spleens of immunized guinea pigs, a result comparable to that achieved by BCG vaccination.

Adult↗