Biomedical subjects

Pratik Singh

Publications and source records attributed to Pratik Singh.

9 recordsLinked to original sources

PubMed2006

Virus-based nanoparticles (VNPs): platform technologies for diagnostic imaging.

Non-invasive imaging holds great promise for the early detection and treatment of human disease. The ability to both detect and follow disease processes or anatomical defects without biopsy, surgery, or other invasive techniques should lead to lower costs and higher quality of life. The use of "smart" nanoparticles, that combine multiple functions of targeting, imaging, and drug delivery, have tremendous potential to increase the sensitivity and specificity of therapies. These will facilitate early detection and reduce adverse side effects of treatment. There are many different classes of nanoparticles in development including dendrimers, liposomes, paramagnetic nanoparticles, and quantum dots, to name just a few. Here we focus on virus-based nanoparticles (VNPs) as platforms for the development of tissue-specific targeting and imaging agents in vivo.

Animals↗

PubMed2006

Characterization of polymorphism displayed by the coat protein mutants of tomato bushy stunt virus.

Expression of full-length and N-terminal deletion mutants of the coat protein (CP) of tomato bushy stunt virus (TBSV) using the recombinant baculovirus system resulted in spontaneously assembled virus-like particles (VLPs). Deletion of the majority of the R-domain sequence of the CP, residues 1-52 (CP-NDelta52) and 1-62 (CP-NDelta62), produced capsids similar to wild-type VLPs. Interestingly, the CP-NDelta62 mutant that retains the last 3 residues of R-domain is capable of forming both the T = 1 and T = 3 particles. However, between the two types of VLPs, formation of the T = 1 capsids appears to be preferred. Another mutant, CP-NDelta72, in which R-domain (residues 1-65) was completely removed but contains most of the beta-annulus and extended arm (betaA) regions exclusively formed T = 1 particles. These results suggest that as few as 3 residues (63-65) of the R-domain, which includes 2 basic amino acids together with the arm (betaA) and beta-annulus regions, may be sufficient for the formation of T = 3 particles. However, anywhere between 4 to 13 residues of the R-domain may be required for proper positioning of betaA and beta-annulus structural elements of the C-type subunits to facilitate an error free assembly of T = 3 capsids.

Amino Acid Sequence↗

PubMed2006

Canine parvovirus-like particles, a novel nanomaterial for tumor targeting.

Specific targeting of tumor cells is an important goal for the design of nanotherapeutics for the treatment of cancer. Recently, viruses have been explored as nano-containers for specific targeting applications, however these systems typically require modification of the virus surface using chemical or genetic means to achieve tumor-specific delivery. Interestingly, there exists a subset of viruses with natural affinity for receptors on tumor cells that could be exploited for nanotechnology applications. For example, the canine parvovirus (CPV) utilizes transferrin receptors (TfRs) for binding and cell entry into canine as well as human cells. TfRs are over-expressed by a variety of tumor cells and are widely being investigated for tumor-targeted drug delivery. We explored whether the natural tropism of CPV to TfRs could be harnessed for targeting tumor cells. Towards this goal, CPV virus-like particles (VLPs) produced by expression of the CPV-VP2 capsid protein in a baculovirus expression system were examined for attachment of small molecules and delivery to tumor cells. Structural modeling suggested that six lysines per VP2 subunit are presumably addressable for bioconjugation on the CPV capsid exterior. Between 45 and 100 of the possible 360 lysines/particle could be routinely derivatized with dye molecules depending on the conjugation conditions. Dye conjugation also demonstrated that the CPV-VLPs could withstand conditions for chemical modification on lysines. Attachment of fluorescent dyes neither impaired binding to the TfRs nor affected internalization of the 26 nm-sized VLPs into several human tumor cell lines. CPV-VLPs therefore exhibit highly favorable characteristics for development as a novel nanomaterial for tumor targeting.

Journal Article↗

PubMed2005

Systemic trafficking of plant virus nanoparticles in mice via the oral route.

The plant virus, cowpea mosaic virus (CPMV), is increasingly being used as a nanoparticle platform for multivalent display of peptides. A growing variety of applications have employed the CPMV display technology including vaccines, antiviral therapeutics, nanoblock chemistry, and materials science. CPMV chimeras can be inexpensively produced from experimentally infected cowpea plants and are completely stable at 37 degrees C and low pH, suggesting that they could be used as edible or mucosally-delivered vaccines or therapeutics. However, the fate of CPMV particles in vivo, or following delivery via the oral route, is unknown. To address this question, we examined CPMV in vitro and in vivo. CPMV was shown to be stable under simulated gastric conditions in vitro. The pattern of localization of CPMV particles to mouse tissues following oral or intravenous dosing was then determined. For several days following oral or intravenous inoculation, CPMV was found in a wide variety of tissues throughout the body, including the spleen, kidney, liver, lung, stomach, small intestine, lymph nodes, brain, and bone marrow. CPMV particles were detected after cardiac perfusion, suggesting that the particles entered the tissues. This pattern was confirmed using methods to specifically detect the viral capsid proteins and the internal viral RNA. The stability of CPMV virions in the gastrointestinal tract followed by their systemic dissemination supports their use as orally bioavailable nanoparticles.

Administration, Oral↗

PubMed2005

Construction and characterization of a fowlpox virus field isolate whose genome lacks reticuloendotheliosis provirus nucleotide sequences.

Fowlpox virus (FWPV) has been isolated from vaccinated chicken flocks during subsequent fowlpox outbreaks that were characterized by a high degree of mortality and significant economic losses. This inability of current vaccines to induce adequate immunity in poultry could be reflective of an antigenic and/or biologic distinctiveness of FWPV field isolates. In this regard, whereas an infectious reticuloendotheliosis virus (REV) provirus is present in the majority of the field viruses' genomes, only remnants of REV long terminal repeats (LTR) have been retained in the DNAs of each vaccine strain. Although it has not been demonstrated whether the partial LTRs can provide an avenue for FWPV to reacquire the REV provirus by homologous recombination, utilizing viruses of which genomes lack any known integrated retroviral sequences could resolve concern over this issue. Therefore, such an entity was created by genetically modifying a recently isolated field strain of FWPV. This selection, in lieu of a commercial vaccine virus, as the progenitor was based on the probability that a virus circulating in the environment would be more antigenically similar to others in this locale and thus might be a better candidate for vaccine development. A comparison in vivo of the pathogenic traits of the parental wild-type field isolate, its genetically modified progeny, and a rescue mutant in whose genome the REV provirus was inserted at its previous location, indicated that elimination of the provirus sequence correlated with reduced virulence. However, even with elimination of the parasitic REV, the modified FWPV was still slightly more invasive than a commercial vaccine virus. Interestingly, both types of attenuated FWPV elicited a similar degree of antibody production in inoculated chickens and afforded them protection against a subsequent challenge by a field virus, the origin of which was temporally and geographically distinct from that of the progenitor strain. Due to its antigenicity being retained despite a decrease in virulence, this REV-less FWPV could potentially be developed as a vaccine against fowlpox.

Animals↗

PubMed2003

Fowlpox virus infection causes a lymphoproliferative response in chickens.

While attenuated fowlpox virus (FPV) strains are widely used for vaccination of chickens and turkeys for prevention of fowlpox, recombinant FPV expressing various foreign genes have been evaluated for their ability to offer protection against various diseases in poultry as well as mammals. Little is known regarding the cell-mediated immune responses to FPV infection. In this study, immune response in chickens infected with a virulent and a vaccine strain of FPV were compared by a lymphoproliferation assay. Interestingly, a lymphoproliferative response was seen during 2-4 weeks post-infection irrespective of the FPV strain used in this study. Analyses of the buffy coat cultures with (35)S-methionine pulse labeling revealed an elevated protein of approximately 48-50 kDa in the culture supernatants. Furthermore, those supernatants could stimulate naive, non-adherent cells of the buffy coat cultures, in a dose dependant manner, suggestive of stimulatory cytokines. FPV, a complex virus presumably stimulates a variety of cytokines in vivo causing a proliferative cellular response. Knowledge of those cytokines or a better understanding of the proliferative responses is pivotal in evaluation of FPV vaccines and in the design of FPV-based recombinant vaccines.

Animals↗

PubMed2003

Reticuloendotheliosis virus sequences within the genomes of field strains of fowlpox virus display variability.

Nine field strains of fowlpox virus (FPV) isolated during a 24-year span from geographically diverse outbreaks of fowlpox in the United States were screened for the presence of reticuloendotheliosis virus (REV) sequences in their genomes by PCR. Each isolate appeared to be heterogeneous in that either a nearly intact provirus or just a 248- or 508-nucleotide fusion of portions of the integrated REV 5' and 3' long terminal repeats (LTRs) was exclusively present at the same genomic site. In contrast, four fowlpox vaccines of FPV origin and three originating from pigeonpox virus were genetically homogeneous in having retained only the 248-bp LTR fusion, whereas two other FPV-based vaccines had only the larger one. These remnants of integrated REV presumably arose during homologous recombination at one of the two regions common to both LTRs or during retroviral excision from the FPV genome. Loss of the provirus appeared to be a natural event because the tripartite population could be detected in a field sample (tracheal lesion). Moreover, the provirus was also readily deleted during propagation of FPV in cultured cells, as evidenced by the detection of truncated LTRs after one passage of a plaque-purified FPV recombinant having a "genetically marked" provirus. However, the deletion mutants did not appear to have a substantial replicative advantage in vitro because even after 55 serial passages the original recombinant FPV was still prevalent. As to the in vivo environment, retention of the REV provirus may confer some benefit to FPV for infection of poultry previously vaccinated against fowlpox.

3' Untranslated Regions↗

PubMed2003

Identification and characterization of fowlpox virus strains using monoclonal antibodies.

The use of 2 monoclonal antibodies (MAbs), P1D9 and P2D4, which recognize different fowlpox virus (FPV) antigens, for the identification and characterization of FPV strains was evaluated. Initially, the MAbs were used in conjunction with a dot blot assay that enabled FPV to be differentiated from the avian herpesvirus, infectious laryngotracheitis virus. Confirmation of the specificity of these MAbs was provided by the demonstration that only FPV antigens were recognized by a combination of both antibodies when used for immunoblotting proteins contained in various avipoxviruses. Later, an antigenic characterization of 11 FPV field isolates, 6 FPV vaccine strains, and 3 pigeonpox virus vaccines was performed by Western blotting with the individual MAbs. Whereas MAb P2D4 consistently recognized a protein with an apparent molecular weight of 60 kD, there was variability in the size of the antigen that was immunoreactive with the other MAb. For example, MAb P1D9 recognized an antigen of apparent molecular weight of 46 kD in all vaccine strains except 2 of FPV origin. In these exceptions, either only a 39-kD or both a 42- and 46-kD protein were immunoreactive. As for the field isolates, a 39-kD antigen was recognized in 8 of them, whereas a 42-kD antigen was detected in the remaining 3. Therefore, the more extensive immunoblotting technique may facilitate FPV strain differentiation, whereas routine diagnosis of fowlpox could be accomplished by using the MAb-based dot blot assay.

Animals↗

PubMedDate not supplied

Accelerated bioorthogonal conjugation: a practical method for the ligation of diverse functional molecules to a polyvalent virus scaffold.

Covalent bond formation to proteins is made difficult by their multiple unprotected functional groups and normally low concentrations. A water-soluble sulfonated bathophenanthroline ligand (2) was used to promote a highly efficient Cu(I)-mediated azide-alkyne cycloaddition (CuAAC) reaction for the chemoselective attachment of biologically relevant molecules to cowpea mosaic virus (CPMV). The ligated substrates included complex sugars, peptides, poly(ethylene oxide) polymers, and the iron carrier protein transferrin, with routine success even for cases that were previously resistant to azide-alkyne coupling using the conventional ligand tris(triazolyl)amine (1). The use of 4-6 equiv of substrate was sufficient to achieve loadings of 60-115 molecules/virion in yields of 60-85%. Although it is sensitive to oxygen, the reliably efficient performance of the Cu.2 system makes it a useful tool for demanding bioconjugation applications.

Carbohydrates↗