Gag P24 Research Articles

Sequence variations in HIV-1 p24 Gag-derived epitopes can alter binding of KIR2DL2 to HLA-C*03:04 and modulate primary natural killer cell function.

The aim of this study was to assess the consequence of sequence variations in HLA-C03:04-presented HIV-1 p24 Gag epitopes on binding of the inhibitory natural killer (NK) cell receptor KIR2DL2 to HLA-C03:04. HIV-1 may possibly evade recognition by KIR+ NK cells through selection of sequence variants that interfere with the interactions of inhibitory killer cell immunoglobulin-like receptors (KIRs) and their target ligands on HIV-1 infected cells. KIR2DL2 is an inhibitory NK cell receptor that binds to a family of HLA-C ligands. Here, we investigated whether HIV-1 encodes for HLA-C03:04-restricted epitopes that alter KIR2DL2 binding. Tapasin-deficient 721.220 cells expressing HLA-C03:04 were pulsed with overlapping peptides (10mers overlapped by nine amino acids, spanning the entire HIV-1 p24 Gag sequence) to identify peptides that stabilized HLA-C expression. The impact that sequence variation in HLA-C03:04-binding HIV-1 epitopes has on KIR2DL2 binding and KIR2DL2+ NK cell function was determined using KIR2DL2-Fc constructs and NK cell degranulation assays. Several novel HLA-C03:04 binding epitopes were identified within the HIV-1 p24 Gag consensus sequence. Three of these consensus sequence peptides (Gag144-152, Gag163-171 and Gag295-304) enabled binding of KIR2DL2 to HLA-C03:04 and resulted in inhibition of KIR2DL2+ primary NK cells. Furthermore, naturally occurring minor variants of epitope Gag295-304 enhanced KIR2DL2 binding to HLA-C03:04. Our data show that naturally occurring sequence variations within HLA-C03:04-restricted HIV-1 p24 Gag epitopes can have a significant impact on the binding of inhibitory KIR receptors and primary NK cell function.

Patterns of recombination in HIV-1M are influenced by selection disfavouring the survival of recombinants with disrupted genomic RNA and protein structures.

Genetic recombination is a major contributor to the ongoing diversification of HIV. It is clearly apparent that across the HIV-genome there are defined recombination hot and cold spots which tend to co-localise both with genomic secondary structures and with either inter-gene boundaries or intra-gene domain boundaries. There is also good evidence that most recombination breakpoints that are detectable within the genes of natural HIV recombinants are likely to be minimally disruptive of intra-protein amino acid contacts and that these breakpoints should therefore have little impact on protein folding. Here we further investigate the impact on patterns of genetic recombination in HIV of selection favouring the maintenance of functional RNA and protein structures. We confirm that chimaeric Gag p24, reverse transcriptase, integrase, gp120 and Nef proteins that are expressed by natural HIV-1 recombinants have significantly lower degrees of predicted folding disruption than randomly generated recombinants. Similarly, we use a novel single-stranded RNA folding disruption test to show that there is significant, albeit weak, evidence that natural HIV recombinants tend to have genomic secondary structures that more closely resemble parental structures than do randomly generated recombinants. These results are consistent with the hypothesis that natural selection has acted both in the short term to purge recombinants with disrupted RNA and protein folds, and in the longer term to modify the genome architecture of HIV to ensure that recombination prone sites correspond with those where recombination will be minimally deleterious.

Transcriptional and posttranscriptional regulation of cytokine gene expression in HIV-1 antigen-specific CD8+ T cells that mediate virus inhibition.

The ability of CD8+ T cells to effectively limit HIV-1 replication and block HIV-1 acquisition is determined by the capacity to rapidly respond to HIV-1 antigens. Understanding both the functional properties and regulation of an effective CD8+ response would enable better evaluation of T cell-directed vaccine strategies and may inform the design of new therapies. We assessed the antigen specificity, cytokine signature, and mechanisms that regulate antiviral gene expression in CD8+ T cells from a cohort of HIV-1-infected virus controllers (VCs) (<5,000 HIV-1 RNA copies/ml and CD4+ lymphocyte counts of >400 cells/μl) capable of soluble inhibition of HIV-1. Gag p24 and Nef CD8+ T cell-specific soluble virus inhibition was common among the VCs and correlated with substantial increases in the abundance of mRNAs encoding the antiviral cytokines macrophage inflammatory proteins MIP-1α, MIP-1αP (CCL3L1), and MIP-1β; granulocyte-macrophage colony-stimulating factor (GM-CSF); lymphotactin (XCL1); tumor necrosis factor receptor superfamily member 9 (TNFRSF9); and gamma interferon (IFN-γ). The induction of several of these mRNAs was driven through a coordinated response of both increased transcription and stabilization of mRNA, which together accounted for the observed increase in mRNA abundance. This coordinated response allows rapid and robust induction of mRNA messages that can enhance the CD8+ T cells' ability to inhibit virus upon antigen encounter. We show that mRNA stability, in addition to transcription, is key in regulating the direct anti-HIV-1 function of antigen-specific memory CD8+ T cells. Regulation at the level of RNA helps enable rapid recall of memory CD8+ T cell effector functions for HIV-1 inhibition. By uncovering and understanding the mechanisms employed by CD8+ T cell subsets with antigen-specific anti-HIV-1 activity, we can identify new strategies for comprehensive identification of other important antiviral genes. This will, in turn, enhance our ability to inhibit virus replication by informing both cure strategies and HIV-1 vaccine designs that aim to reduce transmission and can aid in blocking HIV-1 acquisition.

Induction of CD8+ T-cell responses against subunit antigens by the novel cationic liposomal CAF09 adjuvant

Vaccines inducing cytotoxic T-cell responses are required to achieve protection against cancers and intracellular infections such as HIV and Hepatitis C virus. Induction of CD8+ T cell responses in animal models can be achieved by the use of viral vectors or DNA vaccines but so far without much clinical success. Here we describe the novel CD8+ T-cell inducing adjuvant, cationic adjuvant formulation (CAF) 09, consisting of dimethyldioctadecylammonium (DDA)-liposomes stabilized with monomycoloyl glycerol (MMG)-1 and combined with the TLR3 ligand, Poly(I:C). Different antigens from tuberculosis (TB10.3, H56), HIV (Gag p24), HPV (E7) and the model antigen ovalbumin were formulated with CAF09 and administering these vaccines to mice resulted in a high frequency of antigen-specific CD8+ T cells. CAF09 was superior in its ability to induce antigen-specific CD8+ T cells as compared to other previously described CTL-inducing adjuvants, CAF05 (DDA/trehalose dibehenate (TDB)/Poly(I:C)), Aluminium/monophosphoryl lipid-A (MPL) and Montanide/CpG/IL-2. The optimal effect was obtained when the CAF09-adjuvanted vaccine was administered by the i.p. route, whereas s.c. administration primed limited CD8+ T-cell responses. The CD4+ T cells induced by CAF09 were mainly of an effector-memory-like phenotype and the CD8+ T cells were highly cytotoxic. Finally, in a mouse therapeutic skin tumor model, the HPV-16 E7 antigen formulated in CAF09 significantly reduced the growth of already established subcutaneous E7-expressing TC-1 tumors in 38% of the mice and in a corresponding prophylactic model 100% of the mice were protected. Thus, CAF09 is a potent new adjuvant which is able to induce CD8+ T-cell responses against several antigens and to enhance the protective efficacy of an E7 vaccine both in a therapeutic and in a prophylactic tumor model.

Oral and vaginal epithelial cell lines bind and transfer cell-free infectious HIV-1 to permissive cells but are not productively infected.

The majority of HIV-1 infections worldwide are acquired via mucosal surfaces. However, unlike the vaginal mucosa, the issue of whether the oral mucosa can act as a portal of entry for HIV-1 infection remains controversial. To address potential differences with regard to the fate of HIV-1 after exposure to oral and vaginal epithelium, we utilized two epithelial cell lines representative of buccal (TR146) and pharyngeal (FaDu) sites of the oral cavity and compared them with a cell line derived from vaginal epithelium (A431) in order to determine (i) HIV-1 receptor gene and protein expression, (ii) whether HIV-1 genome integration into epithelial cells occurs, (iii) whether productive viral infection ensues, and (iv) whether infectious virus can be transferred to permissive cells. Using flow cytometry to measure captured virus by HIV-1 gp120 protein detection and western blot to detect HIV-1 p24 gag protein, we demonstrate that buccal, pharyngeal and vaginal epithelial cells capture CXCR4- and CCR5-utilising virus, probably via non-canonical receptors. Both oral and vaginal epithelial cells are able to transfer infectious virus to permissive cells either directly through cell-cell attachment or via transcytosis of HIV-1 across epithelial cells. However, HIV-1 integration, as measured by real-time PCR and presence of early gene mRNA transcripts and de novo protein production were not detected in either epithelial cell type. Importantly, both oral and vaginal epithelial cells were able to support integration and productive infection if HIV-1 entered via the endocytic pathway driven by VSV-G. Our data demonstrate that under normal conditions productive HIV-1 infection of epithelial cells leading to progeny virion production is unlikely, but that epithelial cells can act as mediators of systemic viral dissemination through attachment and transfer of HIV-1 to permissive cells.

Anti-HIV-1 activity of Sargassum swartzii a marine brown alga

Methods The HIV inhibitory activity of aqueous and methanolic extracts of S. swartzii was determined on laboratory adapted strains of HIV-1 clades C and A by indirectly measuring reduction in HIV-1 gag p24 antigen levels and inhibition of HIV-1 RT activity in supernatants of virus infected donor peripheral blood mononuclear cell (PBMC) cultures grown in varying concentrations of the extracts. Phytochemical analysis and cytotoxicity of the extracts was also undertaken.

HIV-1 NEF ligands predominate in the HLA class I of infected CD4+ T cells (APP2P.108)

Abstract Class I HLA reveal virus-derived ligands to cytotoxic T lymphocytes (CTL) whose function is to eliminate infected cells. The Los Alamos National Laboratory (LANL) database reports numerous HIV-1 CTL epitopes, none of which have elicited protective immunity in vaccine testing. The goal of this study was to determine the number and nature of HIV-1 ligands available for CTL targeting through presentation by the HLA class I of virus-infected cells. Class I presented peptides were recovered from immunoaffinity purified HLA-A*11:01 gathered from HIV-1 (NL4-3)-infected human CD4+ SUP-T1 T cells. These peptides were fractionated by RP-HPLC and mapped by tandem mass spectrometry (MS/MS). Seven HIV-derived peptides were confirmed by MS/MS as unique to infected cells. Twelve LANL HIV epitopes, including the well-studied Gag p24 epitope ACQGVGGPGHK (AK11), were clearly absent from the HLA-A*11:01 of infected cells. Of the 7 HIV-1 ligands found to be presented by A*11:01, 4 were derived from Nef, 2 from Gag, and 1 from Pol. When these 7 ligands were tested for T cell reactivity in a gamma interferon ELISPOT assay using PBMC from HIV-1 infected individuals (including elite controllers), the Nef-derived peptides were found to be highly reactive. These data demonstrate that the class I HLA of HIV-1 infected CD4+ T cells present a handful of viral peptide ligands for recognition by CTL and that reported HIV-1 CTL epitopes might not be presented by the HLA class I of infected cells.

Longer sequence insertions in p6 gag play a role in immune escape in HIV-1 subtype C

Methods In a prospective study, a cohort of select seropositive drug naive subjects is being monitored at YRG CARE, Chennai for a period of two years with repeated sampling at 6-month intervals. The viral RNA was extracted from plasma and Gag was amplified, followed by Sanger sequencing. The samples of interest were further subjected to next-generation sequencing using Illumina MiSeq and analyzed using the CLC Genomics Workbench software.

HIV-1 Subtypes B and C Unique Recombinant Forms (URFs) and Transmitted Drug Resistance Identified in the Western Cape Province, South Africa

South Africa has the largest worldwide HIV/AIDS population with 5.6 million people infected and at least 2 million people on antiretroviral therapy. The majority of these infections are caused by HIV-1 subtype C. Using genotyping methods we characterized HIV-1 subtypes of the gag p24 and pol PR and RT fragments, from a cohort of female participants in the Western Cape Province, South Africa. These participants were recruited as part of a study to assess the combined brain and behavioural effects of HIV and early childhood trauma. The partial HIV-1 gag and pol fragments of 84 participants were amplified by PCR and sequenced. Different online tools and manual phylogenetic analysis were used for HIV-1 subtyping. Online tools included: REGA HIV Subtyping tool version 3; Recombinant Identification Program (RIP); Context-based Modeling for Expeditious Typing (COMET); jumping profile Hidden Markov Models (jpHMM) webserver; and subtype classification using evolutionary algorithms (SCUEAL). HIV-1 subtype C predominates within the cohort with a prevalence of 93.8%. We also show, for the first time, the presence of circulating BC strains in at least 4.6% of our study cohort. In addition, we detected transmitted resistance associated mutations in 4.6% of analysed sequences. With tourism and migration rates to South Africa currently very high, we are detecting more and more HIV-1 URFs within our study populations. It is stil unclear what role these unique strains will play in terms of long term antiretroviral treatment and what challenges they will pose to vaccine development. Nevertheless, it remains vitally important to monitor the HIV-1 diversity in South Africa and worldwide as the face of the epidemic is continually changing.

Microbial exposure alters HIV-1-induced mucosal CD4+ T cell death pathways Ex vivo

BackgroundEarly HIV-1 infection causes massive CD4+ T cell death in the gut and translocation of bacteria into the circulation. However, the programmed cell death (PCD) pathways used by HIV-1 to kill CD4+ T cells in the gut, and the impact of microbial exposure on T cell loss, remain unclear. Understanding mucosal HIV-1 triggered PCD could be advanced by an ex vivo system involving lamina propria mononuclear cells (LPMCs). We therefore modeled the interactions of gut LPMCs, CCR5-tropic HIV-1 and a commensal gut bacterial species, Escherichia coli. In this Lamina Propria Aggregate Culture (LPAC) model, LPMCs were infected with HIV-1BaL by spinoculation and cultured in the presence or absence of heat killed E.coli. CD4+ T cell numbers derived from flow cytometry and viable cell counts were reported relative to mock infection. Viable cells were identified by viability dye exclusion (AqVi), and intracellular HIV-1 Gag p24 protein was used to identify infected cells. Annexin V and AqVi were used to identify apoptotic versus necrotic cells. Caspase-1 and Caspase-3 activities were blocked using specific inhibitors YVAD and DEVD, respectively.ResultsCD4+ T cell depletion following HIV-1 infection was reproducibly observed by 6 days post infection (dpi). Depletion at 6 dpi strongly correlated with infection frequency at 4 dpi, was significantly blocked by Efavirenz treatment, and was primarily driven by p24-negative cells that were predominantly necrotic. HIV-1 infection significantly induced CD4+ T-cell intrinsic Caspase-1 activity, whereas Caspase-1 inhibition, but not Caspase-3 inhibition, significantly blocked CD4+ T cell depletion. Exposure to E.coli enhanced HIV-1 infection and CD4+ T depletion, and significantly increased the number of apoptotic p24+ cells. Notably, CD4+ T cell depletion in the presence of E.coli was partially blocked by Caspase-3, but not by Caspase-1 inhibition.ConclusionsIn the LPAC model, HIV-1 induced Caspase-1 mediated pyroptosis in bystander CD4+ T cells, but microbial exposure shifted the PCD mechanism toward apoptosis of productively infected T cells. These results suggest that mucosal CD4+ T cell death pathways may be altered in HIV-infected individuals after gut barrier function is compromised, with potential consequences for mucosal inflammation, viral dissemination and systemic immune activation.

Bacillus subtilis Spores as Vaccine Adjuvants: Further Insights into the Mechanisms of Action

Bacillus subtilis spores have received growing attention regarding potential biotechnological applications, including the use as probiotics and in vaccine formulations. B. subtilis spores have also been shown to behave as particulate vaccine adjuvants, promoting the increase of antibody responses after co-administration with antigens either admixed or adsorbed on the spore surface. In this study, we further evaluated the immune modulatory properties of B. subtilis spores using a recombinant HIV gag p24 protein as a model antigen. The adjuvant effects of B. subtilis spores were not affected by the genetic background of the mouse lineage and did not induce significant inflammatory or deleterious effects after parenteral administration. Our results demonstrated that co-administration, but not adsorption to the spore surface, enhanced the immunogenicity of that target antigen after subcutaneous administration to BALB/c and C57BL/6 mice. Spores promoted activation of antigen presenting cells as demonstrated by the upregulation of MHC and CD40 molecules and enhanced secretion of pro-inflammatory cytokines by murine dendritic cells. In addition, in vivo studies indicated a direct role of the innate immunity on the immunomodulatory properties of B. subtilis spores, as demonstrated by the lack of adjuvant effects on MyD88 and TLR2 knockout mouse strains.

Altered Response Hierarchy and Increased T-Cell Breadth upon HIV-1 Conserved Element DNA Vaccination in Macaques

HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24gag elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55gag increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist.

The phosphorylation of HIV-1 Gag by atypical protein kinase C facilitates viral infectivity by promoting Vpr incorporation into virions

BackgroundHuman immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein that mediates the assembly and release of virus-like particles (VLPs) from an infected cell membrane. The Gag C-terminal p6 domain contains short sequence motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. Gag p6 has also been found to be phosphorylated during HIV-1 infection and this event may affect virus replication. However, the kinase that directs the phosphorylation of Gag p6 toward virus replication remains to be identified. In our present study, we identified this kinase using a proteomic approach and further delineate its role in HIV-1 replication.ResultsA proteomic approach was designed to systematically identify human protein kinases that potently interact with HIV-1 Gag and successfully identified 22 candidates. Among this panel, atypical protein kinase C (aPKC) was found to phosphorylate HIV-1 Gag p6. Subsequent LC-MS/MS and immunoblotting analysis with a phospho-specific antibody confirmed both in vitro and in vivo that aPKC phosphorylates HIV-1 Gag at Ser487. Computer-assisted structural modeling and a subsequent cell-based assay revealed that this phosphorylation event is necessary for the interaction between Gag and Vpr and results in the incorporation of Vpr into virions. Moreover, the inhibition of aPKC activity reduced the Vpr levels in virions and impaired HIV-1 infectivity of human primary macrophages.ConclusionOur current results indicate for the first time that HIV-1 Gag phosphorylation on Ser487 is mediated by aPKC and that this kinase may regulate the incorporation of Vpr into HIV-1 virions and thereby supports virus infectivity. Furthermore, aPKC inhibition efficiently suppresses HIV-1 infectivity in macrophages. aPKC may therefore be an intriguing therapeutic target for HIV-1 infection.

The neutralizing function of the anti-HTLV-1 antibody is essential in preventing in vivo transmission of HTLV-1 to human T cells in NOD-SCID/¿cnull (NOG) mice

Human T-cell leukemia virus type 1 (HTLV-1) causes both neoplastic and inflammatory diseases, including adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Because these life-threatening and disabling diseases are not yet curable, it is important to prevent new HTLV-1 infections. In this study, we have established a simple humanized mouse model of HTLV-1 infection for evaluating prophylactic and therapeutic interventions. In this model, HTLV-1-negative normal human peripheral blood mononuclear cells (PBMCs) are transplanted directly into the spleens of severely immunodeficient NOD-SCID/γcnull (NOG) mice, together with mitomycin-treated HTLV-1-producing T cells. Using this model, we tested the efficacy of monoclonal antibodies (mAbs) specific to HTLV-1 as well as human IgG isolated from HAM/TSP patients (HAM-IgG) in preventing HTLV-1-infection. One hour before and 24 h after transplantation of the human cells, each antibody sample was inoculated intraperitoneally. On day 14, human PBMCs isolated from the mouse spleens were tested for HTLV-1 infection. Whereas fresh CD4-positive and CD8-positive T cells isolated from untreated mice or mice treated with isotype control mAb, HTLV-1 non-neutralizing mAbs to envelope gp46, gag p19, and normal human IgG were all infected with HTLV-1; the mice treated with either HTLV-1 neutralizing anti-gp46 mAb or HAM-IgG did not become infected. Our data indicate that the neutralizing function of the antibody, but not the antigen specificity, is essential for preventing the in vivo transmission of HTLV-1. The present animal model will also be useful for the in vivo evaluation of the efficacy of candidate molecules to be used as prophylactic and therapeutic intervention against HTLV-1 infection.

The effects of opioids on HIV reactivation in latently-infected T-lymphoblasts.

BackgroundOpioids may have effects on susceptibility to HIV-infection, viral replication and disease progression. Injecting drug users (IDU), as well as anyone receiving opioids for anesthesia and analgesia may suffer the clinical consequences of such interactions. There is conflicting data between in vitro experiments showing an enhancing effect of opioids on HIV replication and clinical data, mostly showing no such effect. For clarification we studied the effects of the opioids heroin and morphine on HIV replication in cultured CD4-positive T cells at several concentrations and we related the observed effects with the relevant reached plasma concentrations found in IDUs.MethodsLatently-infected ACH-2 T lymphoblasts were incubated with different concentrations of morphine and heroine. Reactivation of HIV was assessed by intracellular staining of viral Gag p24 protein and subsequent flow cytometric quantification of p24-positive cells. The influence of the opioid antagonist naloxone and the antioxidants N-acetyl-cysteine (NAC) and glutathione (GSH) on HIV reactivation was determined. Cell viability was investigated by 7-AAD staining and flow cytometric quantification.ResultsMorphine and heroine triggered reactivation of HIV replication in ACH-2 cells in a dose-dependent manner at concentrations above 1 mM (EC50 morphine 2.82 mM; EC50 morphine 1.96 mM). Naloxone did not interfere with heroine-mediated HIV reactivation, even at high concentrations (1 mM). Opioids also triggered necrotic cell death at similar concentrations at which HIV reactivation was observed. Both opioid-mediated reactivation of HIV and opioid-triggered cell death could be inhibited by the antioxidants GSH and NAC.ConclusionsOpioids reactivate HIV in vitro but at concentrations that are far above the plasma levels of analgesic regimes or drug concentrations found in IDUs. HIV reactivation was mediated by effects unrelated to opioid-receptor activation and was tightly linked to the cytotoxic activity of the substances at millimolar concentrations, suggesting that opioid-mediated reactivation of HIV was due to accompanying effects of cellular necrosis such as activation of reactive oxygen species and NF-κB.

Complete prevention of HTLV-1 infection in humanized mice (hu-PBL SCID) by a neutralizing monoclonal antibody to envelope gp46

Human T-cell leukemia virus type 1 (HTLV-1) causes both neoplastic and inflammatory diseases: adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Since these disabling and/or life threatening diseases are not yet curable, it is important to prevent new infections. In this study, we have established a simple humanized mouse model of HTLV-1 infection for evaluating therapeutic and immunomodulatory interventions. Using this model, we tested the effect of HTLV-1 specific neutralizing antibodies. HTLV-1-negative normal human peripheral blood mononuclear cells (PBMCs) were transplanted directly into the spleens of severely immunodeficient mice (NOD/SCID/γCnull: NOG) together with the mitomycin-treated HTLV-1 producing T cells (ILT-M1). Before (one hour) and after (24 hours) transplantation of human PBMCs, monoclonal antibodies against HTLV-1 as well as human IgG isolated from both HTLV-1 infected and non-infected individuals were inoculated intraperitonealy. On day 14, human PBMCs were isolated from mouse spleen, and tested for HTLV-1 infection by real time PCR and flow cytometry. Similar to the naturally HTLV-1 infected PBMCs, both CD4+ and CD8+ T cells isolated from untreated or isotype antibody treated mice were found to be HTLV-1 infected, and the CD8+ T cells harbored HTLV-1 to a lesser extent. Also, HTLV-1 Tax expression was negative in isolated human PBMCs but became positive after 16 hours of culture. Although non-neutralizing monoclonal antibodies to gp46, monoclonal antibody to gag p19, and normal human IgG did not block the infection, neutralizing monoclonal antibody to gp46 and human anti-HTLV-1 IgG completely blocked the infection. Our findings provide a new strategy for preventing initial HTLV-1 infection and blocking further spread in vivo. The potential mechanisms involved in the antibody effect will also be discussed.

Safety, Tolerability, and Immunogenicity of Repeated Doses of DermaVir, a Candidate Therapeutic HIV Vaccine, in HIV-Infected Patients Receiving Combination Antiretroviral Therapy

HIV-specific cellular immune responses are associated with control of viremia and delayed disease progression. An effective therapeutic vaccine could mimic these effects and reduce the need for continued antiretroviral therapy. DermaVir, a topically administered plasmid DNA-nanomedicine expressing HIV (CladeB) virus-like particles consisting of 15 antigens, induces predominantly central memory T-cell responses. Treated HIV-infected adults (HIV RNA <50 and CD4 >350) were randomized to placebo or escalating DermaVir doses (0.1 or 0.4 mg of plasmid DNA at weeks 1, 7, and 13 in the low- and intermediate-dose groups and 0.8 mg at weeks 0, 1, 6, 7, 12, and 13 in the high-dose group), n = 5-6 evaluable subjects per group. Immunogenicity was assessed by a 12-day cultured interferon-γ enzyme-linked immunosorbent spot assay at baseline and at weeks 9, 17, and 37 using 1 Tat/Rev and 3 overlapping Gag peptide pools (p17, p24, and p15). Groups were comparable at baseline. The study intervention was well tolerated, without dose-limiting toxicities. Most responses were highest at week 17 (4 weeks after last vaccination) when Gag p24 responses were significantly greater among intermediate-dose group compared with control subjects [median (IQR): 67,600 (5633-74,368) versus 1194 (9-1667)] net spot-forming units per million cells, P = 0.032. In the intermediate-dose group, there was also a marginal Gag p15 response increase from baseline to week 17 [2859 (1867-56,933), P = 0.06], and this change was significantly greater than in the placebo group [0 (-713 to 297), P = 0.016]. DermaVir administration was associated with a trend toward greater HIV-specific, predominantly central memory T-cell responses. The intermediate DermaVir dose tended to show the greatest immunogenicity, consistent with previous studies in different HIV-infected patient populations.

Increased seroreactivity to HERV-K10 peptides in patients with HTLV myelopathy

BackgroundPreviously, we had shown that persons infected with human T-cell lymphoma leukemia virus 1 or 2 (HTLV-1 or 2) had an increased prevalence of antibodies to a peptide in the Pol protein of the retrovirus HERV-K10, homologous to a peptide in HTLV gp21 envelope protein. The prevalence rate was higher in those with myelopathy vs. non-myelopathy. We have now extended our observations to a cohort restricted to North America in whom the diagnosis of HTLV myelopathy was rigorously confirmed to also test for reactivity to another HERV-K10 peptide homologous to the HTLV p24 Gag protein.MethodsSera from 100 volunteer blood donors (VBD), 53 patients with large granular lymphocytic leukemia (LGLL), 74 subjects with HTLV-1 or 2 infection (58 non-myelopathy and 16 myelopathy) and 83 patients with multiple sclerosis (MS) were evaluated in ELISA assays using the above peptides.ResultsThe HTLV myelopathy patients had a statistically significant increased prevalence of antibodies to both HERV-K10 peptides (87.5%) vs. the VBD (0%), LGLL patients (0%), MS patients (4.8%), and the HTLV positive non-myelopathy subjects (5.2%).ConclusionThe data suggest that immuno-cross-reactivity to HERV-K10 peptides and/or transactivation of HERV-K10 expression by the HTLV Tax protein may be involved in the pathogenesis of HTLV-associated myelopathy/tropical spastic paraparesis and spastic ataxia.

Therapeutic DNA Vaccination of Vertically HIV-Infected Children: Report of the First Pediatric Randomised Trial (PEDVAC)

SubjectsTwenty vertically HIV-infected children, 6–16 years of age, with stable viral load control and CD4+ values above 400 cells/mm3.InterventionTen subjects continued their ongoing antiretroviral treatment (ART, Group A) and 10 were immunized with a HIV-DNA vaccine in addition to their previous therapy (ART and vaccine, Group B). The genetic vaccine represented HIV-1 subtypes A, B and C, encoded Env, Rev, Gag and RT and had no additional adjuvant. Immunizations took place at weeks 0, 4 and 12, with a boosting dose at week 36. Monitoring was performed until week 60 and extended to week 96.ResultsSafety data showed good tolerance of the vaccine. Adherence to ART remained high and persistent during the study and did not differ significantly between controls and vaccinees. Neither group experienced either virological failure or a decline of CD4+ counts from baseline. Higher HIV-specific cellular immune responses were noted transiently to Gag but not to other components of the vaccine. Lymphoproliferative responses to a virion antigen HIV-1 MN were higher in the vaccinees than in the controls (p = 0.047), whereas differences in reactivity to clade-specific Gag p24, RT or Env did not reach significance. Compared to baseline, the percentage of HIV-specific CD8+ lymphocytes releasing perforin in the Group B was higher after the vaccination schedule had been completed (p = 0.031). No increased CD8+ perforin levels were observed in control Group A.ConclusionsThe present study demonstrates the feasibility, safety and moderate immunogenicity of genetic vaccination in vertically HIV-infected children, paving the way for amplified immunotherapeutic approaches in the pediatric population.Trial registration clinicaltrialsregister.eu _2007-002359-18 IT

Multiple Independent Introductions of HIV-1 CRF01_AE Identified in China: What Are the Implications for Prevention?

BackgroundHIV-1 CRF01_AE accounts for an important fraction of HIV infections in Asia including China, but little is known about the phylogenetic and evolutionary history of this CRF (circulating recombinant form). In the current study, we collected a large number of 1,957 CRF01_AE gag p17 sequences with known sampling year (1990-2010) from 5 global regions representing 15 countries to better understand the phylogenetic relationships and epidemic history of CRF01_AE strains in China.Methodology/Principal FindingsCRF01_AE gag p17 sequences were retrieved from public databases to explore phylogenetic relationships and phylogeographic dynamics of CRF01_AE in Asia by using maximum-likelihood phylogenetics and Bayesian coalescent-based analyses. We found close phylogenetic relationships between sequences from Thailand, Vietnam and China. Moreover, at least 5 independent introductions and 5 independent autochthonous clades of CRF01_AE, which descended from Thailand or Vietnam were identified in China from 1991 through 2003.Conclusion/SignificanceThe current study not only defines the migration of CRF01_AE clades to/in Asia, but also demonstrates the criticalness of identifying the circulating strains in the population for the development of vaccine and microbicides.