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Biomedical subjects

Bharat Ramratnam

Publications and source records attributed to Bharat Ramratnam.

17 recordsLinked to original sources
PubMed2006

An anti-HIV microbicide engineered in commensal bacteria: secretion of HIV-1 fusion inhibitors by lactobacilli.

OBJECTIVES: To engineer Lactobacillus spp. to secrete HIV-1 fusion inhibitors with potent neutralizing activity against primary HIV-1 isolates. METHODS: HIV-1 fusion inhibitors (FI-1, FI-2, and FI-3) were introduced into the previously developed shuttle vector pTSV2 and transformed in L. plantarum and L. gasseri. The signal peptide Usp45 from L. lactis was used to achieve high secretion efficiency of peptides into the bacterial supernatant. The antiviral activity of lactobacillus-derived HIV-1 fusion inhibitors was tested against a panel of primary HIV-1 isolates and a chimeric simian/HIV (SHIV-162P3) using the TZM infection assay. TZM-bl cells are engineered HeLa cells that express CD4, CCR5, and CXCR4 and contain integrated reporter genes for firefly luciferase and beta-galactosidase under the control of an HIV-1 long terminal repeat. The amount of secreted fusion inhibitor FI-3 was determined by Western blot analysis and the antiviral specificity verified by antibody-mediated depletion of peptide FI-3 and HIV-1 infection with VSV-G envelope pseudotyped virions. RESULTS: Viral infectivity of primary HIV-1 isolates and SHIV-162P3 was neutralized by up to 98% and 72%, respectively, by 10% (v/v) lactobacillus supernatant containing fusion inhibitor FI-3. The antiviral activity of the lactobacillus-derived fusion inhibitor FI-3 was clearly shown to be attributable to the secreted fusion inhibitor peptide. CONCLUSION: The development of recombinant lactobacilli expressing HIV-1 fusion inhibitors with potent neutralizing activity represents an important step toward the development of a live microbial (topical) microbicide against HIV-1 transmission.

Animals↗
PubMed2006

Access to nectin favors herpes simplex virus infection at the apical surface of polarized human epithelial cells.

Viral entry may preferentially occur at the apical or the basolateral surfaces of polarized cells, and differences may impact pathogenesis, preventative strategies, and successful implementation of viral vectors for gene therapy. The objective of these studies was to examine the polarity of herpes simplex virus (HSV) entry using several different human epithelial cell lines. Human uterine (ECC-1), colonic (CaCo-2), and retinal pigment (ARPE-19) epithelial cells were grown on collagen-coated inserts, and the polarity was monitored by measuring the transepithelial cell resistance. Controls were CaSki cells, a human cervical cell line that does not polarize in vitro. The polarized cells, but not CaSki cells, were 16- to 50-fold more susceptible to HSV infection at the apical surface than at the basolateral surface. Disruption of the tight junctions by treatment with EGTA overcame the restriction on basolateral infection but had no impact on apical infection. No differences in binding at the two surfaces were observed. Confocal microscopy demonstrated that nectin-1, the major coreceptor for HSV entry, sorted preferentially to the apical surface, overlapping with adherens and tight junction proteins. Transfection with small interfering RNA specific for nectin-1 resulted in a significant reduction in susceptibility to HSV at the apical surface but had little impact on basolateral infection. Infection from the apical but not the basolateral surface triggered focal adhesion kinase phosphorylation and led to nuclear transport of viral capsids and viral gene expression. These studies indicate that access to nectin-1 contributes to preferential apical infection of these human epithelial cells by HSV.

Active Transport, Cell Nucleus↗
PubMed2006

Engineering mucosal RNA interference in vivo.

Mucosal surfaces serve as a gateway to disease. Here, we demonstrate that RNA interference can be used to manipulate mucosal gene expression in vivo. Using a murine model, we show that direct application of liposome-complexed siRNA mediates gene-specific silencing in cervicovaginal and rectal mucosa. A single vaginal or rectal administration of siRNA targeting hematopoietic or somatic cell gene products reduced corresponding mRNA levels by up to 90%. Using a murine model of inflammatory bowel disease, we found that the rectal application of siRNA targeting TNF-alpha led to relative mucosal resistance to experimental colitis. Liposomal siRNA formulations proved nontoxic, did not elicit a nonspecific interferon response, and provide a means for genetic engineering of mucosal surfaces in vivo.

Animals↗
PubMed2005

Bioengineering lactic acid bacteria to secrete the HIV-1 virucide cyanovirin.

An urgent need exists to prevent the sexual transmission of HIV-1. With prevalence rates exceeding 35% in parts of sub-Saharan Africa, increasing attention has been placed on developing and testing microbicidal agents capable of preventing virus transmission at mucosal sites. HIV-1 microbicides must meet several requirements before their widespread use. The drugs must be able to neutralize a diversity of HIV-1 strains, not induce mucosal inflammation, be associated with minimal side effects, and be effective for a prolonged period after a single application. Recent work has demonstrated the utility of recombinant lactic acid bacteria (LAB) as agents of mucosal drug delivery. Here, we describe the bioengineering of strains of LAB to secrete the prototypic virucidal compound cyanovirin (CV-N) and demonstrate the anti-HIV-1 activity of secreted CV-N. Our results suggest that recombinant LAB may serve as effective microbicidal compounds and deserve in vivo testing in simian immunodeficiency virus models of mucosal virus transmission.

Bacterial Proteins↗
PubMed2005

HIV type 1 genotypic variation in an antiretroviral treatment-naive population in southern India.

Most studies of HIV-1 drug resistance have examined subtype B viruses; fewer data are available from developing countries, where non-B subtypes predominate. We determined the prevalence of mutations at protease and reverse transcriptase drug resistance positions in antiretroviral drug-naive individuals in southern India. The pol region of the genome was amplified from plasma HIV-1 RNA in 50 patients. All sequences clustered with HIV-1 subtype C. All patients had at least one protease and/or RT mutation at a known subtype B drug resistance position. Twenty percent of patients had mutations at major protease inhibitor resistance positions and 100% had mutations at minor protease inhibitor resistance positions. Six percent and 14% of patients had mutations at nucleoside reverse transcriptase inhibitor and/or nonnucleoside reverse transcriptase inhibitor resistance positions, respectively. Larger scale studies need to be undertaken to better define the genotypic variation of circulating Indian subtype C viruses and their potential impact on drug susceptibility and clinical outcome in treated individuals.

Adult↗
PubMed2004

Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins.

Post-transcriptional inhibition of HIV-1 replication can be achieved by RNA interference (RNAi). The cellular expression of short interfering RNA (siRNA) or short hairpin RNA (shRNA) homologous to regions of the HIV-1 genome decreases viral replication by the selective degradation of targeted RNA. Here, we demonstrate that another class of noncoding regulatory RNA, termed microRNA (miRNA), can be used to deliver antiviral RNAi. By incorporating sequences encoding siRNA targeting the HIV-1 transactivator protein tat into a human miR-30 pre-microRNA (pre-miRNA) backbone, we were able to express tat siRNA in cells. The tat siRNA delivered as pre-miRNA precursor was 80% more effective in reducing HIV-1 p24 antigen production than tat siRNA expressed as conventional shRNA. Our results confirm the utility of expressing HIV-1 specific siRNA through a miR-30 precursor stem-loop structure and suggest that this strategy can be used to increase the antiviral potency of RNAi.

Base Sequence↗
PubMed2004

Intensification of antiretroviral therapy accelerates the decay of the HIV-1 latent reservoir and decreases, but does not eliminate, ongoing virus replication.

This study evaluated whether intensification of standard antiretroviral therapy with abacavir, with or without efavirenz, leads to better viral suppression and acceleration of the rate of HIV-1 decay. Ten HIV-1-infected individuals were enrolled in a prospective, open-label study and received standard, combination antiretroviral therapy with either 3 or 4 agents. The rate of decay of the HIV-1 latent reservoir and the frequency of intermittent viremia were compared between 5 patients who underwent treatment intensification and 5 control subjects with comparable baseline characteristics. When compared with control patients, the median half-life (t1/2) of the latent reservoir decreased from 31 to 10 months (P = 0.016) in subjects who had treatment intensification. The frequency of intermittent viremia/year also decreased in 4 of 5 individuals following intensification (2.4/y vs. 0.8/y). These data suggest that ongoing virus replication during standard antiretroviral therapy is due, in part, to the inadequate antiviral potency of current regimens. Despite better viral suppression, treatment intensification did not completely block viral replication, as evidenced by continuing intermittent viremia in some individuals. Additional studies are needed to understand the host- and pathogen-related determinants of incomplete pharmacologic control of HIV-1 replication.

Adult↗
PubMed2004

Efficient gene transfer of HIV-1-specific short hairpin RNA into human lymphocytic cells using recombinant adeno-associated virus vectors.

The cellular introduction of short, interfering RNA leads to sequence-specific degradation of homologous mRNA, a process termed RNA interference (RNAi). Here, we report that recombinant adeno-associated virus 2 (rAAV-2) can be used to transfer short hairpin (sh) RNA expression cassettes genetically into human cells. HIV-1 replication was suppressed by >95% in H9 cells and primary human lymphocytes that expressed shRNA targeting the first exon of the viral transactivator protein tat compared to control cells. rAAV-2 integrated stably into the host genome, leading to long-term expression of tat shRNA. Our findings demonstrate the utility of rAAV-2 for the genetic transfer of shRNA expression cassettes into human cells, providing an alternative to using retroviral vectors as RNAi delivery systems.

Blotting, Northern↗
PubMed2004

HIV-1-specific RNA interference.

The preclinical development of RNA interference (RNAi) as a novel therapeutic agent for HIV-1 infection is reviewed. RNAi refers to the sequence-specific degradation of RNA that follows the cellular introduction of homologous, short-interfering RNA (siRNA). RNAi has emerged as a powerful tool to probe the function of genes of known sequence in vitro and in vivo. Advances in vector design permit the effective expression of siRNA in human cells by transfer of short hairpin RNA expression cassettes. Recent investigations have described the ability of RNAi to decrease the replication of HIV-1 in lymphocytic cells using siRNA targeting viral (eg, Tat, Gag and Rev) and host (eg, CCR5 and CD4) proteins. Can RNAi be used as a form of genetic therapy for HIV-1 and associated infections? There are numerous challenges associated with converting RNAi from a laboratory technique to an antiviral therapeutic. Recent research on the cellular delivery, antiviral durability and gene-silencing specificity of HIV-1-specific RNAi is reviewed.

Genetic Therapy↗
PubMed2003

Nucleotide sequence homology requirements of HIV-1-specific short hairpin RNA.

The degradation of a selected mRNA species by RNA interference requires a high degree of homology between the short interfering or short hairpin RNA (si or shRNA) and its target. Recent reports have demonstrated that the number and location of nucleotide mismatches affect the activity of si/shRNA. Here, we systematically examined the effect of single nucleotide mutations in all 21 positions of an effective shRNA that targets the gag gene of HIV-1. We found that all mutant shRNAs exerted RNAi activity but were less effective in gene silencing compared to the wild-type gag shRNA. The most pronounced reduction in function was observed with mutations in the central and 5' regions of the shRNA. Our results demonstrate that optimal gene silencing requires perfect homology between shRNA and the chosen target, but that a variable degree of silencing occurs, depending upon the precise location of nucleotide mismatches.

Base Sequence↗
PubMed2003

Promoter choice affects the potency of HIV-1 specific RNA interference.

RNA interference (RNAi) is mediated by small interfering (si) RNAs that target and degrade mRNA in a sequence-specific manner. Cellular expression of siRNA can be achieved by the use of expression cassettes driven by RNA polymerase III (pol III) promoters. Here, we demonstrate that a modified tRNA(met)-derived (MTD) promoter effectively drives the cellular expression of HIV-1-specific siRNA. We observed up to 56% greater inhibition of virus production when the MTD promoter was used to drive the expression of short hairpin (sh) RNA targeting the HIV-1 transactivator protein tat compared to cassettes containing other pol III promoters such as H1, U6+1 and U6+27. We conclude that the MTD promoter is ideally suited to drive intracellular expression of HIV-1 specific siRNA and may serve as an important component of future RNAi vector delivery systems.

Cell Line↗
PubMed2003

Human immunodeficiency virus type 1 escape from RNA interference.

Sequence-specific degradation of mRNA by short interfering RNA (siRNA) allows the selective inhibition of viral proteins that are critical for human immunodeficiency virus type 1 (HIV-1) replication. The aim of this study was to characterize the potency and durability of virus-specific RNA interference (RNAi) in cell lines that stably express short hairpin RNA (shRNA) targeting the HIV-1 transactivator protein gene tat. We found that the antiviral activity of tat shRNA was abolished due to the emergence of viral quasispecies harboring a point mutation in the shRNA target region. Our results suggest that, in order for RNAi to durably suppress HIV-1 replication, it may be necessary to target highly conserved regions of the viral genome. Alternatively, similar to present antiviral drug therapy paradigms, DNA constructs expressing multiple siRNAs need to be developed that target different regions of the viral genome, thereby reducing the probability of generating escape mutants.

Cell Line↗
PubMed2002

Discontinuation of antiretroviral therapy commenced early during the course of human immunodeficiency virus type 1 infection, with or without adjunctive vaccination.

Sixteen subjects were treated with highly active antiretroviral therapy within 120 days of the onset of symptoms of newly acquired human immunodeficiency virus type 1 (HIV-1) infection. Eleven of the 16 participated in an adjunctive therapeutic vaccine trial. After a mean of 3.2 years of treatment, they elected to discontinue therapy. Virus rebound occurred in all subjects and was followed by a spontaneous, transient although significant reduction in log plasma HIV-1 RNA level, ranging from 0.3 to 3.1 log(10) copies/mL. Despite evidence of the induction of HIV-1-specific cell-mediated immune responses, plasma viremia was not persistently suppressed to <500 copies/mL in any subject. The magnitude and dynamics of virus rebound were similar in both vaccinated and unvaccinated subjects. Nevertheless, given the transient suppression of viremia observed in nearly all subjects after treatment has been discontinued, further investigations of adjunctive vaccination with optimized antiretroviral therapy in treating HIV-1 infection are warranted.

AIDS Vaccines↗
PubMed2002

Evolving patterns of HIV-1 resistance to antiretroviral agents in newly infected individuals.

OBJECTIVE: To assess temporal changes in prevalence of transmitted HIV-1 drug resistance in a homogeneous cohort of newly infected individuals. METHODS: Pretreatment genotypic and phenotypic drug resistance was tested in 154 subjects with primary HIV-1 infection identified between 1995 and 2001 (group A; n = 76) and 1999 and 2001 (group B; n = 78). Sequence analysis was assessed by population-based sequencing. Virus susceptibility to antiretroviral agents was determined by the PhenoSense assay (ViroLogic). RESULTS: The frequency of resistance-associated mutations in protease (PR) and reverse transcriptase (RT) genes increased from 13.2% (1995-1998) to 19.7% (1999-2001). Although the overall prevalence of viruses with phenotypic resistance did not vary (1995-1998, 10.0%; 1999-2001, 10.8%), the distribution of drug classes changed [nucleoside RT inhibitor (NRTI): 8.3% to 2.7%; non-NRTI: 5.0% to 8.1%; protease inhibitors (PI): 1.7% to 5.4%]. The decrease of phenotypic resistance to NRTI in 1999-2001 was caused by the absence of transmitted lamivudine-resistant variants. Phenotypically susceptible variants with aspartic acid or serine residues at position 215 of RT (5.3%; P = 0.04) instead emerged. Hypersusceptibility to PI decreased from 18.3% to 5.4% (P = 0.02) while the amino acid substitutions in PR increased over time: M36I (6.6% to 19.7%) and A71V/T (3.9% to 15.8%). CONCLUSIONS: There was an increase in the number of HIV-1 variants with both genotypic and phenotypic resistance to non-NRTI and PI over time. Furthermore, viruses with altered genotypes compatible with thymidine analogue or PI exposure but susceptible phenotypes were seen in 1999-2001. The latter findings suggest transmission of viruses from subjects who have either changed or discontinued therapy.

Adult↗
PubMed2002

Residual viral replication during antiretroviral therapy boosts human immunodeficiency virus type 1-specific CD8+ T-cell responses in subjects treated early after infection.

Human immunodeficiency virus type 1 (HIV-1)-infected subjects treated early after infection have preserved HIV-1-specific CD4+ T-cell function. We studied the effect of highly active antiretroviral therapy (HAART) on the frequency of HIV-1-specific CD8+ T cells in patients treated during early (n = 31) or chronic (n = 23) infection. The degree of viral suppression and time of initiation of treatment influenced the magnitude of the CD8+ T-cell response. HIV-1-specific CD8+ T cells can increase in number after HAART in subjects treated early after infection who have episodes of transient viremia.

Acute Disease↗