Dinesh K. Singh

Biodegradable nanoparticles are excellent vehicle for site directed in-vivo delivery of drugs and vaccines

Biodegradable nanoparticles (NPs) are gaining increased attention for their ability to serve as a viable carrier for site specific delivery of vaccines, genes, drugs and other biomolecules in the body. They offer enhanced biocompatibility, superior drug/vaccine encapsulation, and convenient release profiles for a number of drugs, vaccines and biomolecules to be used in a variety of applications in the field of medicine. In this manuscript, the methods of preparation of biodegradable NPs, different factors affecting optimal drug encapsulation, factors affecting drug release rates, various surface modifications of nanoparticles to enhance in-vivo circulation, distribution and multimodal functionalities along with the specific applications such as tumor targeting, oral delivery, and delivery of these particles to the central nervous system have been reviewed.

Silver nanoparticles are broad-spectrum bactericidal and virucidal compounds

The advance in nanotechnology has enabled us to utilize particles in the size of the nanoscale. This has created new therapeutic horizons, and in the case of silver, the currently available data only reveals the surface of the potential benefits and the wide range of applications. Interactions between viral biomolecules and silver nanoparticles suggest that the use of nanosystems may contribute importantly for the enhancement of current prevention of infection and antiviral therapies. Recently, it has been suggested that silver nanoparticles (AgNPs) bind with external membrane of lipid enveloped virus to prevent the infection. Nevertheless, the interaction of AgNPs with viruses is a largely unexplored field. AgNPs has been studied particularly on HIV where it was demonstrated the mechanism of antiviral action of the nanoparticles as well as the inhibition the transmission of HIV-1 infection in human cervix organ culture. This review discusses recent advances in the understanding of the biocidal mechanisms of action of silver Nanoparticles.

Use of silver nanoparticles increased inhibition of cell-associated HIV-1 infection by neutralizing antibodies developed against HIV-1 envelope proteins

BackgroundHIV/AIDS pandemic is a worldwide public health issue. There is a need for new approaches to develop new antiviral compounds or other therapeutic strategies to limit viral transmission. The envelope glycoproteins gp120 and gp41 of HIV are the main targets for both silver nanoparticles (AgNPs) and neutralizing antibodies. There is an urgency to optimize the efficiency of the neutralizing antibodies (NABs). In this study, we demonstrated that there is an additive effect between the four NABs and AgNPs when combined against cell-associated HIV-1 infection in vitroResultsFour NABs (Monoclonal antibody to HIV-1 gp41 126-7, HIV-1 gp120 Antiserum PB1 Sub 2, HIV-1 gp120 Antiserum PB1, HIV-1 gp120 Monoclonal Antibody F425 B4e8) with or without AgNPs of 30-50 nm in size were tested against cell free and cell-associated HIVIIIB virus. All NABs inhibited HIV-1 cell free infection at a dose response manner, but with AgNPs an antiviral additive effect was not achieved Although there was no inhibition of infection with cell-associated virus by the NABs itself, AgNPs alone were able to inhibit cell associated virus infection and more importantly, when mixed together with NABs they inhibited the HIV-1 cell associated infection in an additive manner.DiscussionThe most attractive strategies to deal with the HIV problem are the development of a prophylactic vaccine and the development of effective topical vaginal microbicide. For two decades a potent vaccine that inhibits transmission of infection of HIV has been searched. There are vaccines that elicit NABs but none of them has the efficacy to stop transmission of HIV-1 infection. We propose that with the addition of AgNPs, NABs will have an additive effect and become more potent to inhibit cell-associated HIV-1 transmission/infection.ConclusionsThe addition of AgNPs to NABs has significantly increased the neutralizing potency of NABs in prevention of cell-associated HIV-1 transmission/infection. Further exploration is required to standardize potentiation of NABs by AgNPs. It is also required to evaluate in vivo toxicity of AgNPs before AgNPs could be incorporated in any antiviral vaginal creams.

Sequence Note: Comparison of Vpu Sequences from Diverse Geographical Isolates of HIV Type 1 Identifies the Presence of Highly Variable Domains, Additional Invariant Amino Acids, and a Signature Sequence Motif Common to Subtype C Isolates

We compared the Vpu sequences from 101 strains of HIV-1 isolated from diverse geographical regions and various subtypes in order to identify regions of high variability, and those amino acid residues that were highly conserved or invariant. In addition to the highly conserved casein kinase II (CKII) phosphorylation sites, our analysis identified additional invariant residues in the transmembrane domain and in the first and second alpha-helical domains. Our analysis revealed that all subtype C sequences had a conserved LRLL motif at the C terminus that was also found in A/C intersubtype recombinants. While our analysis demonstrated the conservation of CKII domains in HIV-1 group M and O isolates, the number of potential CKII phosphorylation sites was variable in SIVcpz sequences. The results of this study will provide a basis for future mutagenesis studies to examine the role of certain amino acid residues in the structure and function of Vpu.

The presence of the casein kinase II phosphorylation sites of Vpu enhances the CD4+ T cell loss caused by the simian–human immunodeficiency virus SHIVKU-lbMC33 in pig-tailed macaques

The simian-human immunodeficiency virus (SHIV)/ macaque model for human immunodeficiency virus type 1 has become a useful tool to assess the role of Vpu in lentivirus pathogenesis. In this report, we have mutated the two phosphorylated serine residues of the HIV-1 Vpu to glycine residues and have reconstructed a SHIV expressing this nonphosphorylated Vpu (SHIV(S52,56G)). Expression studies revealed that this protein was localized to the same intracellular compartment as wild-type Vpu. To determine if this virus was pathogenic, four pig-tailed macaques were inoculated with SHIV(S52,56G) and virus burdens and circulating CD4(+) T cells monitored up to 1 year. Our results indicate that SHIV(S52,56G) caused rapid loss in the circulating CD4(+) T cells within 3 weeks of inoculation in one macaque (CC8X), while the other three macaques developed no or gradual numbers of CD4(+) T cells and a wasting syndrome. Histological examination of tissues revealed that macaque CC8X had lesions in lymphoid tissues (spleen, lymph nodes, and thymus) that were typical for macaques inoculated with pathogenic parental SHIV(KU-1bMC33) and had no lesions within the CNS. To rule out that macaque CC8X had selected for a virus in which there was reversion of the glycine residues at positions 52 and 56 to serine residues and/or compensating mutations occurred in other genes associated with CD4 down-regulation, sequence analysis was performed on amplified vpu sequences isolated from PBMC and from several lymphoid tissues at necropsy. Sequence analysis revealed a reversion of the glycine residues back to serine residues in this macaque. The other macaques maintained low virus burdens, with one macaque (P003) developing a wasting syndrome between months 9 and 11. Histological examination of tissues from this macaque revealed a thymus with severe atrophy that was similar to that of a previously reported macaque inoculated with a SHIV lacking vpu (Virology 293, 2002, 252). Sequence analysis revealed no reversion of the glycine residues in the vpu sequences isolated from this macaque. These results contrast with those from four macaques inoculated with the parental pathogenic SHIV(KU-1bMC33), all of which developed severe CD4(+) T cell loss within 1 month after inoculation. Taken together, these results indicate that casein kinase II phosphorylation sites of Vpu contributes to the pathogenicity of the SHIV(KU-1bMC33) and suggest that the SHIV(KU-1bMC33)/pig-tailed macaque model will be useful in analyzing amino acids/domains of Vpu that contribute to the pathogenesis of HIV-1.

Association of platelet-derived growth factor-B chain with simian human immunodeficiency virus encephalitis.

Chemokines and cytokines play a critical role in HIV infection, serving both to modulate virus replication and to recruit target cells to the site of infection. Platelet-derived growth factor (PDGF), a mitogen and chemoattractant for a wide variety of cells, is secreted by macrophages. Since macrophages are the target cells for lentiviral infection in the brain and PDGF is a known inducer of macrophage chemoattractant protein-1 (MCP)-1, a potent chemokine closely associated with HIV encephalitis, we investigated the association of PDGF-B chain (PDGF-B) with encephalitis in macaques caused by simian human immunodeficiency virus (SHIV), a chimera of HIV and SIV. Northern blot analysis confirmed elevated expression of PDGF-B chain mRNA in the brains from encephalitic macaques. Validation of these in vivo studies was confirmed in rhesus macrophage cultures infected with SHIV(KU2) in which we demonstrated heightened expression of PDGF-B chain mRNA. Nuclear run-off analysis established transcriptional up-regulation of PDGF-B chain in virus-inoculated macrophage cultures. Reciprocally, addition of exogenous PDGF enhanced virus replication and MCP-1 expression in these cells. Inhibition of virus replication by tyrosine kinase inhibitor, STI-571, and by PDGF-B antisense oligonucleotides confirmed the specificity of the PDGF effect. Relevance of these findings was confirmed by analysis of archival brain tissue from SHIV encephalitic and non-encephalitic macaques for PDGF-B chain expression. PDGF-B chain protein expression was observed in the virus-infected cells in microglial nodules in the brains of SHIV-encephalitic macaques.

A Noninfectious Simian/Human Immunodeficiency Virus DNA Vaccine That Protects Macaques against AIDS

ABSTRACT Simian/human immunodeficiency virus SHIVKU2 replicates with extremely high titers in macaques. In order to determine whether the DNA of the viral genome could be used as a vaccine if the DNA were rendered noninfectious, we deleted the reverse transcriptase gene from SHIVKU2 and inserted this DNA (ΔrtSHIVKU2) into a plasmid that was then used to test gene expression and immunogenicity. Transfection of Jurkat and human embryonic kidney epithelial (HEK 293) cells with the DNA resulted in production of all of the major viral proteins and their precursors and transient export of a large quantity of the Gag p27 into the supernatant fluid. As expected, no infectious virus was produced in these cultures. Four macaques were injected intradermally with 2 mg of the DNA at 0, 8, and 18 weeks. The animals developed neutralizing antibodies and low enzyme-linked immunospot assay (E-SPOT) titers against SHIVKU2. These four animals and two unvaccinated control animals were then challenged with heterologous SHIV89.6P administered into their rectums. The two control animals developed viral RNA titers exceeding 106 copies/ml of plasma, and these titers were accompanied by the loss of CD4+ T cells by 2 weeks after challenge. The two control animals died at weeks 8 and 16, respectively. All four of the immunized animals became infected with the challenge virus but developed lower titers of viral RNA in plasma than the control animals, and the titers decreased over time in three of the four macaques. The fourth animal remained viremic and died at week 47. Whereas the control animals failed to develop E-SPOT responses, all four of the immunized animals developed anamnestic E-SPOT responses after challenge. The animal that died developed the highest E-SPOT response and was the only one that produced neutralizing antibodies against the challenge virus. These results established that noninfectious DNA of pathogenic SHIV could be used as a vaccine to prevent AIDS, even though the immunological assays used did not predict the manner in which the challenge virus would replicate in the vaccinated animals.

Small Ruminant Lentiviruses (SRLVs) Break the Species Barrier to Acquire New Host Range

Zoonotic events of simian immunodeficiency virus (SIV) from non-human primates to humans have generated the acquired immunodeficiency syndrome (AIDS), one of the most devastating infectious disease of the last century with more than 30 million people dead and about 40.3 million people currently infected worldwide. Human immunodeficiency virus (HIV-1 and HIV-2), the two major viruses that cause AIDS in humans are retroviruses of the lentivirus genus. The genus includes arthritis-encephalitis virus (CAEV) and Maedi-Visna virus (MVV), and a heterogeneous group of viruses known as small ruminant lentiviruses (SRLVs), affecting goat and sheep. Lentivirus genome integrates into the host DNA, causing persistent infection associated with a remarkable diversity during viral replication. Direct evidence of mixed infections with these two closely related SRLVs was found in both sheep and goats. The evidence of a genetic continuum with caprine and ovine field isolates demonstrates the absence of an efficient species barrier preventing cross-species transmission. In dual-infected animals, persistent infections with both CAEV and MVV have been described, and viral chimeras have been detected. This not only complicates animal trade between countries but favors the risk that highly pathogenic variants may emerge as has already been observed in the past in Iceland and, more recently, in outbreaks with virulent strains in Spain. SRLVs affecting wildlife have already been identified, demonstrating the existence of emergent viruses adapted to new hosts. Viruses adapted to wildlife ruminants may acquire novel biopathological properties which may endanger not only the new host species but also domestic ruminants and humans. SRLVs infecting sheep and goats follow a genomic evolution similar to that observed in HIV or in other lentiviruses. Lentivirus genetic diversity and host factors leading to the establishment of naturally occurring virulent versus avirulent infections, in addition to the emergence of new strains, challenge every aspect of SRLV control measures for providing efficient tools to prevent the transmission of diseases between wild ungulates and livestock.

Ovine lentivirus-infected macrophages mediate productive infection in cell types that are not susceptible to infection with cell-free virus

Ovine lentiviruses and caprine arthritis-encephalitis virus (CAEV) are prototypic lentiviruses that replicate predominantly in macrophages of infected animals. In situ hybridization of pathologically affected tissues from diseased animals has shown that viral RNA exists in permissive macrophages as well as in non-macrophage cell types that do not support productive virus replication. These findings raise questions about the cellular tropism of these viruses in vivo and how this may relate to their pathogenesis and the establishment of persistent infections. In this study, the susceptibility of macrophages and fibro-epithelial cells derived from goat synovial membrane (GSM) to infection by 14 North American ovine lentivirus strains was examined. All 14 strains were macrophage-tropic, as indicated by expression of viral proteins and by fusion and development of syncytial cytopathic effects following co-culture of infected macrophages with GSM cells. In contrast, neither viral DNA nor viral proteins was detected in GSM cells inoculated with cell-free virus from nine of the 14 strains. Specific virus proteins were immunoprecipitated from restrictive GSM cells following culture with infected macrophages and serial passage of GSM cells to remove the macrophages. The lack of infection of GSM cells by cell-free virus from some ovine lentivirus field strains was circumvented by cell-associated virus infection from infected macrophages to GSM cells following cell-to-cell contact. This strategy could be one of the mechanisms involved in the escape from immune surveillance and establishment of persistent infection in infected animals.

Protection Against Late-Onset AIDS in Macaques Prophylactically Immunized with a Live Simian HIV Vaccine Was Dependent on Persistence of the Vaccine Virus

This is a 5-year follow-up study on 12 macaques that were immunized orally with two live SHIV vaccines, six with V1 and six with V2. All 12 macaques became persistently infected after transient replication of the vaccine viruses; all were challenged vaginally 6 mo later with homologous pathogenic SHIVKU-1. Two of the V1 group developed full-blown AIDS without evidence of vaccine virus DNA in tissues. The data on the 10 vaccinated survivors showed that all 10 became infected with SHIVKU-1 and that DNA of both vaccine and SHIVKU-1 viruses were present 6 mo postchallenge, with minimal replication of SHIVKU-1. During the following 5 years, these animals remained persistently infected, but with only one of the two viruses. Six animals eliminated their vaccine virus after variable periods of time and four of these succumbed to reactivation of the challenge virus and AIDS. Five years after challenge, four latently infected animals, two with V2 and two with SHIVKU-1, were reinoculated with SHIVKU-1. This resulted in transient superinfection and the animals promptly returned to their prechallenge status. Immunosuppression of the four animals 1 year later with Abs to CD8+ lymphocytes resulted in transiently productive replication of their respective latent viruses, and upon recovery of CD8+ lymphocytes, they reverted to their latent virus status. The major finding was that of eight animals that eliminated the vaccine virus, six developed AIDS. The two others harboring SHIVKU-1 remain at risk for developing late-onset disease. The primary correlate against AIDS was persistence of the vaccine virus.