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Find video protocols related to scientific articles indexed in Pubmed.
Antibody to the gp120 V1/V2 Loops and CD4+ and CD8+ T Cell Responses in Protection from SIVmac251 Vaginal Acquisition and Persistent Viremia.
J. Immunol.
PUBLISHED: 11-16-2014
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The human papillomavirus pseudovirions (HPV-PsVs) approach is an effective gene-delivery system that can prime or boost an immune response in the vaginal tract of nonhuman primates and mice. Intravaginal vaccination with HPV-PsVs expressing SIV genes, combined with an i.m. gp120 protein injection, induced humoral and cellular SIV-specific responses in macaques. Priming systemic immune responses with i.m. immunization with ALVAC-SIV vaccines, followed by intravaginal HPV-PsV-SIV/gp120 boosting, expanded and/or recruited T cells in the female genital tract. Using a stringent repeated low-dose intravaginal challenge with the highly pathogenic SIVmac251, we show that although these regimens did not demonstrate significant protection from virus acquisition, they provided control of viremia in a number of animals. High-avidity Ab responses to the envelope gp120 V1/V2 region correlated with delayed SIVmac251 acquisition, whereas virus levels in mucosal tissues were inversely correlated with antienvelope CD4(+) T cell responses. CD8(+) T cell depletion in animals with controlled viremia caused an increase in tissue virus load in some animals, suggesting a role for CD8(+) T cells in virus control. This study highlights the importance of CD8(+) cells and antienvelope CD4(+) T cells in curtailing virus replication and antienvelope V1/V2 Abs in preventing SIVmac251 acquisition.
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Co-dependence of HTLV-1 p12 and p8 Functions in Virus Persistence.
PLoS Pathog.
PUBLISHED: 11-01-2014
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HTLV-1 orf-I is linked to immune evasion, viral replication and persistence. Examining the orf-I sequence of 160 HTLV-1-infected individuals; we found polymorphism of orf-I that alters the relative amounts of p12 and its cleavage product p8. Three groups were identified on the basis of p12 and p8 expression: predominantly p12, predominantly p8 and balanced expression of p12 and p8. We found a significant association between balanced expression of p12 and p8 with high viral DNA loads, a correlate of disease development. To determine the individual roles of p12 and p8 in viral persistence, we constructed infectious molecular clones expressing p12 and p8 (D26), predominantly p12 (G29S) or predominantly p8 (N26). As we previously showed, cells expressing N26 had a higher level of virus transmission in vitro. However, when inoculated into Rhesus macaques, cells producing N26 virus caused only a partial seroconversion in 3 of 4 animals and only 1 of those animals was HTLV-1 DNA positive by PCR. None of the animals exposed to G29S virus seroconverted or had detectable viral DNA. In contrast, 3 of 4 animals exposed to D26 virus seroconverted and were HTLV-1 positive by PCR. In vitro studies in THP-1 cells suggested that expression of p8 was sufficient for productive infection of monocytes. Since orf-I plays a role in T-cell activation and recognition; we compared the CTL response elicited by CD4+ T-cells infected with the different HTLV-1 clones. Although supernatant p19 levels and viral DNA loads for all four infected lines were similar, a significant difference in Tax-specific HLA.A2-restricted killing was observed. Cells infected with Orf-I-knockout virus (12KO), G29S or N26 were killed by CTLs, whereas cells infected with D26 virus were resistant to CTL killing. These results indicate that efficient viral persistence and spread require the combined functions of p12 and p8.
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Multiple Low-dose Challenges in a Rhesus Macaque AIDS Vaccine Trial Result in an Evolving Host Response that affects Protective Outcome.
Clin. Vaccine Immunol.
PUBLISHED: 10-03-2014
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Using whole-blood transcriptional profiling, we investigated differences in the host response to vaccination and challenge in a rhesus macaque AIDS vaccine trial. Samples were collected from animals prior to and after vaccination with live irradiated vaccine cells secreting the modified endoplasmic reticulum chaperone gp96-Ig loaded with simian immunodeficiency virus (SIV) peptides, either alone or in combination with a SIV-gp120 protein boost. Additional samples were collected following multiple low-dose rectal challenge with SIVmac251. Animals in the boosted group had a 73% reduced risk of infection. Surprisingly, few changes in gene expression were observed during the vaccination phase. Focusing on post-challenge comparisons, in particular for protected animals, we identified a host response signature of protection comprised of strong interferon signaling after the first challenge, which then largely abated after further challenges. We also identified a host response signature, comprised of early macrophage-mediated inflammatory responses, in animals with undetectable viral load five days after the first challenge, but which had unusually high viral titers after subsequent challenges. Statistical analysis showed that prime-boost vaccination significantly lowered the probability of infection in a time-consistent manner throughout several challenges. Given that humoral responses in the prime-boost group were highly significant pre-challenge correlates of protection, the strong innate signaling after the first challenge suggests that interferon signaling could possibly enhance vaccine-induced antibody responses and is an important contributor to protection from infection during repeated low-dose exposure to SIV.
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Comparative analysis of SIV-specific cellular immune responses induced by different vaccine platforms in rhesus macaques.
Clin. Immunol.
PUBLISHED: 06-13-2014
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To identify the most promising vaccine candidates for combinatorial strategies, we compared five SIV vaccine platforms including recombinant canary pox virus ALVAC, replication-competent adenovirus type 5 host range mutant RepAd, DNA, modified vaccinia Ankara (MVA), peptides and protein in distinct combinations. Three regimens used viral vectors (prime or boost) and two regimens used plasmid DNA. Analysis at necropsy showed that the DNA-based vaccine regimens elicited significantly higher cellular responses against Gag and Env than any of the other vaccine platforms. The T cell responses induced by most vaccine regimens disseminated systemically into secondary lymphoid tissues (lymph nodes, spleen) and effector anatomical sites (including liver, vaginal tissue), indicative of their role in viral containment at the portal of entry. The cellular and reported humoral immune response data suggest that combination of DNA and viral vectors elicits a balanced immunity with strong and durable responses able to disseminate into relevant mucosal sites.
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Enhanced In Vitro Transcytosis of Simian Immunodeficiency Virus Mediated by Vaccine-Induced Antibody Predicts Transmitted/Founder Strain Number After Rectal Challenge.
J. Infect. Dis.
PUBLISHED: 05-21-2014
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?The time to acquisition of simian immunodeficiency virus (SIV) infection following low-dose repeated rectal challenge correlated inversely with the number of transmitted/founder strains among macaques vaccinated with ALVAC-SIV/gp120 or gp120 alone. We determined if the ability of postvaccination, prechallenge sera to enhance SIVmac251 transcytosis across epithelial cells was associated with transmitted/founder strain number.
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Vaccine-induced myeloid cell population dampens protective immunity to SIV.
J. Clin. Invest.
PUBLISHED: 05-16-2014
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Vaccines are largely evaluated for their ability to promote adaptive immunity, with little focus on the induction of negative immune regulators. Adjuvants facilitate and enhance vaccine-induced immune responses and have been explored for mediating protection against HIV. Using a regimen of peptide priming followed by a modified vaccinia Ankara (MVA) boost in a nonhuman primate model, we found that an SIV vaccine incorporating molecular adjuvants mediated partial protection against rectal SIVmac251 challenges. Animals treated with vaccine and multiple adjuvants exhibited a reduced viral load (VL) compared with those treated with vaccine only. Surprisingly, animals treated with adjuvant alone had reduced VLs that were comparable to or better than those of the vaccine-treated group. VL reduction was greatest in animals with the MHC class I allele Mamu-A*01 that were treated with adjuvant only and was largely dependent on CD8+ T cells. Early VLs correlated with Ki67+CCR5+CD4+ T cell frequency, while set-point VL was associated with expansion of a myeloid cell population that was phenotypically similar to myeloid-derived suppressor cells (MDSCs) and that suppressed T cell responses in vitro. MDSC expansion occurred in animals receiving vaccine and was not observed in the adjuvant-only group. Collectively, these results indicate that vaccine-induced MDSCs inhibit protective cellular immunity and suggest that preventing MDSC induction may be critical for effective AIDS vaccination.
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Humoral immunity induced by mucosal and/or systemic SIV-specific vaccine platforms suggests novel combinatorial approaches for enhancing responses.
Clin. Immunol.
PUBLISHED: 02-18-2014
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Combinatorial HIV/SIV vaccine approaches targeting multiple arms of the immune system might improve protective efficacy. We compared SIV-specific humoral immunity induced in rhesus macaques by five vaccine regimens. Systemic regimens included ALVAC-SIVenv priming and Env boosting (ALVAC/Env); DNA immunization; and DNA plus Env co-immunization (DNA&Env). RepAd/Env combined mucosal replication-competent Ad-env priming with systemic Env boosting. A Peptide/Env regimen, given solely intrarectally, included HIV/SIV peptides followed by MVA-env and Env boosts. Serum antibodies mediating neutralizing, phagocytic and ADCC activities were induced by ALVAC/Env, RepAd/Env and DNA&Env vaccines. Memory B cells and plasma cells were maintained in the bone marrow. RepAd/Env vaccination induced early SIV-specific IgA in rectal secretions before Env boosting, although mucosal IgA and IgG responses were readily detected at necropsy in ALVAC/Env, RepAd/Env, DNA&Env and DNA vaccinated animals. Our results suggest that combined RepAd priming with ALVAC/Env or DNA&Env regimen boosting might induce potent, functional, long-lasting systemic and mucosal SIV-specific antibodies.
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Antiretroviral therapy partly reverses the systemic and mucosal distribution of NK cell subsets that is altered by SIVmac??? infection of macaques.
Virology
PUBLISHED: 01-21-2014
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We characterized three subsets of NK cells in blood, and two subsets in mucosal tissues. SIVmac251 infection increased total and CD16(+) NK cells in the blood. In the rectum, we observed a significant increase in total and NKG2A(+) NK cells during SIV infection. In contrast, the NKp44(+) subset significantly depleted in acute infection and continued to decline in frequency during chronic phase. During SIV infection, blood CD16 and mucosal NKG2A(+) subsets had increased cytotoxic potential. Intriguingly, the NKp44(+) NK cell subtype that likely mediates mucosal homeostasis via the production of cytokines, acquired cytotoxicity. Antiretroviral therapy significantly increased the frequency of mucosal NKG2A(+) NK cells and peripheral CD16(+) NK cells. However, it failed to restore the normal frequency of NKp44(+) NK cells in the rectum. Thus, SIVmac251 infection causes changes in the distribution and function of NK cells and antiretroviral therapy during chronic infection only partially restores NK homeostasis and function.
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Glucocorticoid Treatment at Moderate Doses of SIVmac251-Infected Rhesus Macaques Decreases the Frequency of Circulating CD14(+)CD16(++) Monocytes But Does Not Alter the Tissue Virus Reservoir.
AIDS Res. Hum. Retroviruses
PUBLISHED: 01-18-2014
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Abstract Subsets of CD16-positive monocytes produce proinflammatory cytokines and expand during chronic infection with the human immunodeficiency virus type 1 (HIV). HIV-infected macrophage in tissues may be long lived and contribute to the establishment and maintenance of the HIV reservoir. We found that the (intermediate) CD14(++)CD16(+) and (nonclassical) CD14(+)CD16(++) monocyte subsets are significantly expanded during infection of Rhesus macaques with pathogenic SIVmac251 but not during infection of sooty mangabeys with the nonpathogenic isolate SIVSM. In vitro glucocorticoid (GC) treatment of peripheral blood mononuclear cells (PBMCs) from uninfected or SIVmac251-infected Rhesus macaques and HIV-infected patients treated or not with antiretroviral therapy (ART) resulted in a significant decrease in the frequency of both CD16-positive monocyte subsets. Short-term in vivo treatment with high doses of GC of chronically SIVmac251-infected macaques resulted in a significant decrease in the CD14(+)CD16(++) population and, to a lesser extent, in the CD14(++)CD16(+) monocytes, as well as a significant decrease in the number of macrophages in tissues. Surprisingly, treatment of SIVmac251-infected macaques with ART significantly increased the CD14(++)CD16(+) population and the addition of GC resulted in a significant decrease in only the CD14(+)CD16(++) subset. No difference in SIV DNA levels in blood, lymph nodes, gut, and spleen was found between the groups treated with ART or ART plus GC. Thus, it appears that high doses of GC treatment in the absence of ART could affect both CD16-positive populations in vivo. Whether the efficacy of this treatment at higher doses to decrease virus levels outweighs its risks remains to be determined.
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Transient Increase of Interferon-Stimulated Genes and No Clinical Benefit by Chloroquine Treatment During Acute Simian Immunodeficiency Virus Infection of Macaques.
AIDS Res. Hum. Retroviruses
PUBLISHED: 12-24-2013
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Abstract Simian immunodeficiency virus (SIV) infection leads to AIDS in experimentally infected Rhesus macaques similarly to HIV-infected humans. In contrast, SIV infection of natural hosts is characterized by a down-regulation of innate acute responses to the virus within a few weeks of infection and results in limited pathology. Chloroquine (CQ) has been used in the treatment or prevention of malaria and has recently been shown to cause a decrease of immune activation and CD4 cell loss in HIV-infected individuals treated with antiretroviral therapy. Here, we treated Rhesus macaques with CQ during the acute phase of SIVmac251 infection with the intent to decrease viral-induced immune activation and possibly limit disease progression. Contrary to what was expected, CQ treatment resulted in a temporary increased expression of interferon (IFN)-stimulating genes and it worsened the recovery of CD4(+) T cells in the blood. Our findings confirm recent results observed in asymptomatic HIV-infected patients and suggest that CQ does not provide an obvious benefit in the absence of antiretroviral therapy.
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Palmitoylation and p8-mediated HTLV-1 transmission.
J. Virol.
PUBLISHED: 11-27-2013
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Orf-I gene of HTLV-1 encodes p8 and p12 and has a conserved cysteine at position 39. p8 and p12 form disulfide-linked dimers and only the monomeric form of p8 or p12 are palmitoylated. Mutation of cysteine 39 to alanine (C39A) abrogated dimerization and palmitoylation of both proteins. However, the ability of p8 to localize to the cell surface and to increase cell adhesion and viral transmission was not affected by the C39A mutation.
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The Canarypox Vector ALVAC Induces Distinct Cytokine Responses Compared to the Vaccinia-Based Vectors MVA and NYVAC in Rhesus Monkeys.
J. Virol.
PUBLISHED: 11-20-2013
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Despite the growing use of poxviral vectors as vaccine candidates for multiple pathogens and cancers, their innate stimulatory properties remain poorly characterized. Here we show that the canarypox-based vector ALVAC induced distinct systemic proinflammatory and antiviral cytokine and chemokine levels following vaccination of rhesus monkeys compared to the vaccinia-based vectors MVA and NYVAC. These data suggest that there are substantial biological differences among leading poxviral vaccine vectors that may influence resultant adaptive immune responses following vaccination.
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Human T-Cell Leukemia/Lymphoma Virus Type 1 p30, but Not p12/p8, Counteracts Toll-Like Receptor 3 (TLR3) and TLR4 Signaling in Human Monocytes and Dendritic Cells.
J. Virol.
PUBLISHED: 10-23-2013
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The human T-cell leukemia/lymphoma virus type 1 (HTLV-1) p30 protein, essential for virus infectivity in vivo, is required for efficient infection of human dendritic cells (DCs) but not B and T cells in vitro. We used a human monocytic cell line, THP-1, and dendritic cells to study the mechanism of p30 and p12/p8 requirements in these cell types. p30 inhibited the expression of interferon (IFN)-responsive genes (ISG) following stimulation by lipopolysaccharide (LPS) of Toll-like receptor 4 (TLR4) and by poly(I·C) of TLR3 but not of TLR7/8 with imiquimod. Results with THP-1 mirrored those for ex vivo human primary monocytes and monocyte-derived dendritic cells (Mo-mDC). The effect of p30 on TLR signaling was also demonstrated by ablating its expression within a molecular clone of HTLV-1. HTLV-1 infection of monocytes inhibited TLR3- and TLR4-induced ISG expression by 50 to 90% depending on the genes, whereas the isogenic clone p30 knockout virus was less effective at inhibiting TLR3 and TRL4 signaling and displayed lower infectivity. Viral expression and inhibition of ISG transcription was, however, rescued by restoration of p30 expression. A chromatin immunoprecipitation assay demonstrated that p30 inhibits initiation and elongation of PU.1-dependent transcription of IFN-?1, IFN-?, and TLR4 genes upon TLR stimulation. In contrast, experiments conducted with p12/p8 did not demonstrate an effect on ISG expression. These results provide a mechanistic explanation of the requirement of p30 for HTLV-1 infectivity in vivo, suggest that dampening interferon responses in monocytes and DCs is specific for p30, and represent an essential early step for permissive HTLV-1 infection and persistence.
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Cytotoxic capacity of SIV-specific CD8(+) T cells against primary autologous targets correlates with immune control in SIV-infected rhesus macaques.
PLoS Pathog.
PUBLISHED: 02-28-2013
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Although the study of non-human primates has resulted in important advances for understanding HIV-specific immunity, a clear correlate of immune control over simian immunodeficiency virus (SIV) replication has not been found to date. In this study, CD8(+) T-cell cytotoxic capacity was examined to determine whether this function is a correlate of immune control in the rhesus macaque (RM) SIV infection model as has been suggested in chronic HIV infection. SIVmac251-infected human reverse transcriptase (hTERT)-transduced CD4(+) T-cell clone targets were co-incubated with autologous macaque effector cells to measure infected CD4(+) T-cell elimination (ICE). Twenty-three SIV-infected rhesus macaques with widely varying plasma viral RNA levels were evaluated in a blinded fashion. Nineteen of 23 subjects (83%) were correctly classified as long-term nonprogressor/elite controller (LTNP/EC), slow progressor, progressor or SIV-negative rhesus macaques based on measurements of ICE (weighted Kappa 0.75). LTNP/EC had higher median ICE than progressors (67.3% [22.0-91.7%] vs. 23.7% [0.0-58.0%], p?=?0.002). In addition, significant correlations between ICE and viral load (r?=?-0.57, p?=?0.01), and between granzyme B delivery and ICE (r?=?0.89, p<0.001) were observed. Furthermore, the CD8(+) T cells of LTNP/EC exhibited higher per-cell cytotoxic capacity than those of progressors (p?=?0.004). These findings support that greater lytic granule loading of virus-specific CD8(+) T cells and efficient delivery of active granzyme B to SIV-infected targets are associated with superior control of SIV infection in rhesus macaques, consistent with observations of HIV infection in humans. Therefore, such measurements appear to represent a correlate of control of viral replication in chronic SIV infection and their role as predictors of immunologic control in the vaccine setting should be evaluated.
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Cutting edge: novel vaccination modality provides significant protection against mucosal infection by highly pathogenic simian immunodeficiency virus.
J. Immunol.
PUBLISHED: 02-11-2013
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Vaccine-induced protection against infection by HIV or highly pathogenic and virulent SIV strains has been limited. In a proof-of-concept study, we show that a novel vaccine approach significantly protects rhesus macaques from mucosal infection by the highly pathogenic strain SIVmac251. We vaccinated three cohorts of 12 macaques each with live, irradiated vaccine cells secreting the modified endoplasmic reticulum chaperone gp96-Ig. Cohort 1 was vaccinated with cells secreting gp96(SIV)Ig carrying SIV peptides. In addition, Cohort 2 received recombinant envelope protein SIV-gp120. Cohort 3 was injected with cells secreting gp96-Ig (no SIV Ags) vaccines. Cohort 2 was protected from infection. After seven rectal challenges with highly pathogenic SIVmac251, the hazard ratio was 0.27, corresponding to a highly significant, 73% reduced risk for viral acquisition. The apparent success of the novel vaccine modality recommends further study.
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Protection afforded by an HIV vaccine candidate in macaques depends on the dose of SIVmac251 at challenge exposure.
J. Virol.
PUBLISHED: 01-16-2013
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We used the simian immunodeficiency virus mac251 (SIV(mac251)) macaque model to study the effect of the dose of mucosal exposure on vaccine efficacy. We immunized macaques with a DNA prime followed by SIV gp120 protein immunization with ALVAC-SIV and gp120 in alum, and we challenged them with SIV(mac251) at either a single high dose or at two repeated low-dose exposures to a 10-fold-lower dose. Infection was neither prevented nor modified following a single high-dose challenge of the immunized macaques. However, two exposures to a 10-fold-lower dose resulted in protection from SIV(mac251) acquisition in 3 out of 12 macaques. The remaining animals that were infected had a modulated pathogenesis, significant downregulation of interferon responsive genes, and upregulation of genes involved in B- and T-cell responses. Thus, the choice of the experimental model greatly influences the vaccine efficacy of vaccines for human immunodeficiency virus (HIV).
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Antibodies to gp120 and PD-1 expression on virus-specific CD8+ T cells in protection from simian AIDS.
J. Virol.
PUBLISHED: 01-16-2013
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We compared the relative efficacies against simian immunodeficiency virus (SIV) challenge of three vaccine regimens that elicited similar frequencies of SIV-specific CD4(+) and CD8(+) T-cell responses but differed in the level of antibody responses to the gp120 envelope protein. All macaques were primed with DNA plasmids expressing SIV gag, pol, env, and Retanef genes and were boosted with recombinant modified vaccinia Ankara virus (MVA) expressing the same genes, either once (1 × MVA) or twice (2 × MVA), or were boosted once with MVA followed by a single boost with replication-competent adenovirus (Ad) type 5 host range mutant (Ad5 h) expressing SIV gag and nef genes but not Retanef or env (1 × MVA/Ad5). While two of the vaccine regimens (1 × MVA and 1 × MVA/Ad5) protected from high levels of SIV replication only during the acute phase of infection, the 2 × MVA regimen, with the highest anti-SIV gp120 titers, protected during the acute phase and transiently during the chronic phase of infection. Mamu-A*01 macaques of this third group exhibited persistent Gag CD8(+)CM9(+) effector memory T cells with low expression of surface Programmed death-1 (PD-1) receptor and high levels of expression of genes associated with major histocompatibility complex class I (MHC-I) and MHC-II antigen. The fact that control of SIV replication was associated with both high titers of antibodies to the SIV envelope protein and durable effector SIV-specific CD8(+) T cells suggests the hypothesis that the presence of antibodies at the time of challenge may increase innate immune recruiting activity by enhancing antigen uptake and may result in improvement of the quality and potency of secondary SIV-specific CD8(+) T-cell responses.
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Superior T memory stem cell persistence supports long-lived T cell memory.
J. Clin. Invest.
PUBLISHED: 01-02-2013
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Long-lived memory T cells are able to persist in the host in the absence of antigen; however, the mechanism by which they are maintained is not well understood. Recently, a subset of human T cells, stem cell memory T cells (TSCM cells), was shown to be self-renewing and multipotent, thereby providing a potential reservoir for T cell memory throughout life. However, their in vivo dynamics and homeostasis still remain to be defined due to the lack of suitable animal models. We identified T cells with a TSCM phenotype and stem cell-like properties in nonhuman primates. These cells were the least-differentiated memory subset, were functionally distinct from conventional memory cells, and served as precursors of central memory. Antigen-specific TSCM cells preferentially localized to LNs and were virtually absent from mucosal surfaces. They were generated in the acute phase of viral infection, preferentially survived in comparison with all other memory cells following elimination of antigen, and stably persisted for the long term. Thus, one mechanism for maintenance of long-term T cell memory derives from the unique homeostatic properties of TSCM cells. Vaccination strategies designed to elicit durable cellular immunity should target the generation of TSCM cells.
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Targeting the vaginal mucosa with human papillomavirus pseudovirion vaccines delivering simian immunodeficiency virus DNA.
J. Immunol.
PUBLISHED: 12-14-2011
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The majority of HIV infections occur via mucosal transmission. Vaccines that induce memory T and B cells in the female genital tract may prevent the establishment and systemic dissemination of HIV. We tested the immunogenicity of a vaccine that uses human papillomavirus (HPV)-based gene transfer vectors, also called pseudovirions (PsVs), to deliver SIV genes to the vaginal epithelium. Our findings demonstrate that this vaccine platform induces gene expression in the genital tract in both cynomolgus and rhesus macaques. Intravaginal vaccination with HPV16, HPV45, and HPV58 PsVs delivering SIV Gag DNA induced Gag-specific Abs in serum and the vaginal tract, and T cell responses in blood, vaginal mucosa, and draining lymph nodes that rapidly expanded following intravaginal exposure to SIV(mac251.) HPV PsV-based vehicles are immunogenic, which warrant further testing as vaccine candidates for HIV and may provide a useful model to evaluate the benefits and risks of inducing high levels of SIV-specific immune responses at mucosal sites prior to SIV infection.
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Fatal pancreatitis in simian immunodeficiency virus SIV(mac251)-infected macaques treated with 2,3-dideoxyinosine and stavudine following cytotoxic-T-lymphocyte-associated antigen 4 and indoleamine 2,3-dioxygenase blockade.
J. Virol.
PUBLISHED: 10-19-2011
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Human immunodeficiency virus (HIV) infection is associated with immune activation, CD4?-T-cell loss, and a progressive decline of immune functions. Antiretroviral therapy (ART) only partially reverses HIV-associated immune dysfunction, suggesting that approaches that target immune activation and improve virus-specific immune responses may be needed. We performed a preclinical study in rhesus macaques infected with the pathogenic simian immunodeficiency virus SIV(mac251) and treated with ART. We tested whether vaccination administered together with cytotoxic-T-lymphocyte-associated antigen 4 (CTLA-4) blockade and treatment with the indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyl-D-tryptophan (D-1mT), decreased immune activation and improved vaccine efficacy. The treatment did not augment vaccine immunogenicity; rather, it dramatically increased ART-related toxicity, causing all treated animals to succumb to acute pancreatitis and hyperglycemic coma. The onset of fulminant diabetes was associated with severe lymphocyte infiltration of the pancreas and complete loss of the islets of Langerhans. Thus, caution should be used when considering approaches aimed at targeting immune activation during ART.
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TRIM5? does not affect simian immunodeficiency virus SIV(mac251) replication in vaccinated or unvaccinated Indian rhesus macaques following intrarectal challenge exposure.
J. Virol.
PUBLISHED: 09-14-2011
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TRIM5? is a natural resistance factor that binds retroviral capsid proteins and restricts virus replication. The B30.2/SPRY domain of TRIM5? is polymorphic in rhesus macaques, and some alleles are associated with reduced simian immunodeficiency virus (SIV) SIV(mac251) and SIV(smE543) replication in vivo. We determined the distribution of TRIM5? alleles by PCR and sequence analysis of the B30.2/SPRY domain in a cohort of 82 macaques. Thirty-nine of these macaques were mock vaccinated, 43 were vaccinated with either DNA-SIV/ALVAC-SIV/gp120, ALVAC-SIV/gp120, or gp120 alone, and all were exposed intrarectally to SIV(mac251) at one of three doses. We assessed whether the TRIM5? genotype of the macaques affected the replication of challenge virus by studying the number of SIV variants transmitted, the number of exposures required, the SIV(mac251) viral level in plasma and tissue, and the CD4(+) T-cell counts. Our results demonstrated that TRIM5? alleles, previously identified as restrictive for SIV(mac251) replication in vivo following intravenous exposure, did not affect SIV(mac251) replication following mucosal exposure, regardless of prior vaccination, challenge dose, or the presence of the protective major histocompatibility complex alleles (MamuA01(+), MamuB08(+), or MamuB017(+)). The TRIM5? genotype had no apparent effect on the number of transmitted variants or the number of challenge exposures necessary to infect the animals. DNA sequencing of the SIV(mac251) Gag gene of the two stocks used in our study revealed SIV(mac239)-like sequences that are predicted to be resistant to TRIM5? restriction. Thus, the TRIM5? genotype does not confound results of mucosal infection of rhesus macaques with SIV(mac251).
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Major histocompatibility complex class II transactivator CIITA is a viral restriction factor that targets human T-cell lymphotropic virus type 1 Tax-1 function and inhibits viral replication.
J. Virol.
PUBLISHED: 08-03-2011
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Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of an aggressive malignancy of CD4+ T lymphocytes. Since the viral transactivator Tax-1 is a major player in T-cell transformation, targeting Tax-1 protein is regarded as a possible strategy to arrest viral replication and to counteract neoplastic transformation. We demonstrate that CIITA, the master regulator of major histocompatibility complex class II gene transcription, inhibits HTLV-1 replication by blocking the transactivating function of Tax-1 both when exogenously transfected in 293T cells and when endogenously expressed by a subset of U937 promonocytic cells. Tax-1 and CIITA physically interact in vivo via the first 108 amino acids of Tax-1 and two CIITA adjacent regions (amino acids 1 to 252 and 253 to 410). Interestingly, only CIITA 1-252 mediated Tax-1 inhibition, in agreement with the fact that CIITA residues from positions 64 to 124 were required to block Tax-1 transactivation. CIITA inhibitory action on Tax-1 correlated with the nuclear localization of CIITA and was independent of the transcription factor NF-YB, previously involved in CIITA-mediated inhibition of Tax-2 of HTLV-2. Instead, CIITA severely impaired the physical and functional interaction of Tax-1 with the cellular coactivators p300/CBP-associated factor (PCAF), cyclic AMP-responsive element binding protein (CREB), and activating transcription factor 1 (ATF1), which are required for the optimal activation of HTLV-1 promoter. Accordingly, the overexpression of PCAF, CREB, and ATF1 restored Tax-1-dependent transactivation of the viral long-terminal-repeat promoter inhibited by CIITA. These findings strongly support our original observation that CIITA, beside increasing the antigen-presenting function for pathogen antigens, acts as an endogenous restriction factor against human retroviruses by blocking virus replication and spreading.
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Vaccine induced antibodies to the first variable loop of human immunodeficiency virus type 1 gp120, mediate antibody-dependent virus inhibition in macaques.
Vaccine
PUBLISHED: 07-08-2011
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The role of antibodies directed against the hyper variable envelope region V1 of human immunodeficiency virus type 1 (HIV-1), has not been thoroughly studied. We show that a vaccine able to elicit strain-specific non-neutralizing antibodies to this region of gp120 is associated with control of highly pathogenic chimeric SHIV(89.6P) replication in rhesus macaques. The vaccinated animal that had the highest titers of antibodies to the amino terminus portion of V1, prior to challenge, had secondary antibody responses that mediated cell killing by antibody-dependent cellular cytotoxicity (ADCC), as early as 2 weeks after infection and inhibited viral replication by antibody-dependent cell-mediated virus inhibition (ADCVI), by 4 weeks after infection. There was a significant inverse correlation between virus level and binding antibody titers to the envelope protein, (R=-0.83, p=0.015), and ADCVI (R=-0.84 p=0.044). Genotyping of plasma virus demonstrated in vivo selection of three SHIV(89.6P) variants with changes in potential N-linked glycosylation sites in V1. We found a significant inverse correlation between virus levels and titers of antibodies that mediated ADCVI against all the identified V1 virus variants. A significant inverse correlation was also found between neutralizing antibody titers to SHIV(89.6) and virus levels (R=-0.72 p=0.0050). However, passive inoculation of purified immunoglobulin from animal M316, the macaque that best controlled virus, to a naïve macaque, resulted in a low serum neutralizing antibodies and low ADCVI activity that failed to protect from SHIV(89.6P) challenge. Collectively, while our data suggest that anti-envelope antibodies with neutralizing and non-neutralizing Fc(R-dependent activities may be important in the control of SHIV replication, they also demonstrate that low levels of these antibodies alone are not sufficient to protect from infection.
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Suppression of HTLV-1 replication by Tax-mediated rerouting of the p13 viral protein to nuclear speckles.
Blood
PUBLISHED: 06-15-2011
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Disease development in human T-cell leukemia virus type 1 (HTLV-1)-infected individuals is positively correlated with the level of integrated viral DNA in T cells. HTLV-1 replication is positively regulated by Tax and Rex and negatively regulated by the p30 and HBZ proteins. In the present study, we demonstrate that HTLV-1 encodes another negative regulator of virus expression, the p13 protein. Expressed separately, p13 localizes to the mitochondria, whereas in the presence of Tax, part of it is ubiquitinated, stabilized, and rerouted to the nuclear speckles. The p13 protein directly binds Tax, decreases Tax binding to the CBP/p300 transcriptional coactivator, and, by reducing Tax transcriptional activity, suppresses viral expression. Because Tax stabilizes its own repressor, these findings suggest that HTLV-1 has evolved a complex mechanism to control its own replication. Further, these results highlight the importance of studying the function of the HTLV-1 viral proteins, not only in isolation, but also in the context of full viral replication.
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TLR agonists and/or IL-15 adjuvanted mucosal SIV vaccine reduced gut CD4? memory T cell loss in SIVmac251-challenged rhesus macaques.
Vaccine
PUBLISHED: 06-06-2011
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Adjuvant plays an important role in increasing and directing vaccine-induced immune responses. In a previous study, we found that a mucosal SIV vaccine using a combination of IL-15 and TLR agonists as adjuvant mediated partial protection against SIVmac251 rectal challenge, whereas neither IL-15 nor TLR agonists alone as an adjuvant impacted the plasma viral loads. In this study, dissociation of CD4(+) T cell preservation with viral loads was observed in the animals vaccinated with adjuvants. Significantly higher levels of memory CD4(+) T cell numbers were preserved after SIVmac251 infection in the colons of the animals vaccinated with vaccine containing any of these adjuvants compared to no adjuvant. When we measured the viral-specific CD8(+) tetramer responses in the colon lamina propria, we found significantly higher levels of gag, tat, and pol epitope tetramer(+) T cell responses in these animals compared to ones without adjuvant, even if some of the animals had similarly high viral loads. Furthermore, this CD4(+) T preservation was positively correlated with increased levels of gag and Tat, but not pol tetramer(+) T cell responses, and inversely correlated with beta-chemokine expression. The pre-challenged APOBEC3G expression level, which has previously been shown inversely associated with viral loads, was further found positively correlated with CD4(+) T cell number preservation. Overall, these data highlight one unrecognized role of adjuvant in HIV vaccine development, and show that vaccines can produce a surprising discordance between CD4(+) T cell levels and SIV viral load.
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Interleukin-21 administration to rhesus macaques chronically infected with simian immunodeficiency virus increases cytotoxic effector molecules in T cells and NK cells and enhances B cell function without increasing immune activation or viral replication.
Vaccine
PUBLISHED: 05-31-2011
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We have previously shown that interleukin-21, a pleiotropic C ?-chain signaling cytokine, induces the expression of the cytotoxic molecules granzyme B (GrB) and perforin in vitro in CD8 T cells and NK cells of chronically HIV infected individuals. In this pilot study, four chronically SIV infected rhesus macaques (RM) in late-stage disease were given two doses of recombinant MamuIL-21, 50 ?g/kg, intravenously 7 days apart, followed by one subcutaneous dose, 100 ?g/kg, 23 days after the second dose. Three animals served as controls. After each dose of IL-21, increases were noted in frequency and mean fluorescence intensity of GrB and perforin expression in memory and effector subsets of CD8 T cells in peripheral blood (PB), in peripheral and mesenteric lymph node (LN) cells, in PB memory and effector CD4 T cells and in NK cells. Frequencies of SIV-gag specific CD107a(+)IFN-?(+) CD8 T cells increased 3.8-fold in PB and 1.8-fold in LN. In addition, PB CD27(+) memory B cells were 2-fold higher and serum SIV antibodies increased significantly after IL-21 administration. No changes were observed in markers of T cell activation, T cell proliferation or plasma virus load. Thus, administration of IL-21 to chronically SIV infected viremic animals was safe, well tolerated and could augment the cytotoxic potential of T cells and NK cells, promote B cell differentiation with increases in SIV antibody titers without discernable increase in cellular activation. Further studies are warranted to elucidate the effects and potential benefit of IL-21 administration in the context of SIV/HIV infection and in SIV/HIV vaccine design.
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Orf-I and orf-II-encoded proteins in HTLV-1 infection and persistence.
Viruses
PUBLISHED: 04-13-2011
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The 3 end of the human T-cell leukemia/lymphoma virus type-1 (HTLV-1) genome contains four overlapping open reading frames (ORF) that encode regulatory proteins. Here, we review current knowledge of HTLV-1 orf-I and orf-II protein products. Singly spliced mRNA from orf-I encodes p12, which can be proteolytically cleaved to generate p8, while differential splicing of mRNA from orf-II results in production of p13 and p30. These proteins have been demonstrated to modulate transcription, apoptosis, host cell activation and proliferation, virus infectivity and transmission, and host immune responses. Though these proteins are not essential for virus replication in vitro, p8, p12, p13, and p30 have an important role in the establishment and maintenance of HTLV-1 infection in vivo.
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Smallpox vaccine safety is dependent on T cells and not B cells.
J. Infect. Dis.
PUBLISHED: 04-01-2011
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The licensed smallpox vaccine, ACAM2000, is a cell culture derivative of Dryvax. Both ACAM2000 and Dryvax are administered by skin scarification and can cause progressive vaccinia, with skin lesions that disseminate to distal sites. We have investigated the immunologic basis of the containment of vaccinia in the skin with the goal to identify safer vaccines for smallpox. Macaques were depleted systemically of T or B cells and vaccinated with either Dryvax or an attenuated vaccinia vaccine, LC16m8. B cell depletion did not affect the size of skin lesions induced by either vaccine. However, while depletion of both CD4(+) and CD8(+) T cells had no adverse effects on LC16m8-vaccinated animals, it caused progressive vaccinia in macaques immunized with Dryvax. As both Dryvax and LC16m8 vaccines protect healthy macaques from a lethal monkeypox intravenous challenge, our data identify LC16m8 as a safer and effective alternative to ACAM2000 and Dryvax vaccines for immunocompromised individuals.
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Gp96 SIV Ig immunization induces potent polyepitope specific, multifunctional memory responses in rectal and vaginal mucosa.
Vaccine
PUBLISHED: 01-13-2011
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The ER-resident chaperone gp96, when released by cell lysis, induces an immunogenic chemokine signature and causes innate immune activation of DC and NK cells. Here we show that intraperitoneal immunization with a genetically engineered, secreted form of gp96, gp96-Ig chaperoning SIV antigens, induces high levels of antigen specific CD8 CTL in the rectal and vaginal mucosa of Rhesus macaques. The frequency of SIV Gag- and SIV Tat-tetramer positive CD8 CTL in the intestinal mucosa reached 30-50% after the third immunization. Tetramer positive CD8 CTL expressed appropriate functional (granzyme B) and migration markers (CD103). The polyepitope specificity of the mucosal CD8 and CD4 response is evident from a strong, multifunctional cytokine response upon stimulation with peptides covering the gag, tat and env proteins. Induction of powerful mucosal effector CD8 CTL responses by cell-based gp96(SIV)-Ig immunization may provide a pathway to the development of safe and effective SIV/HIV vaccines.
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Human T-cell leukemia virus type 1 p8 protein increases cellular conduits and virus transmission.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 11-12-2010
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The human T-cell leukemia virus type 1 (HTLV-1) is the cause of adult T-cell leukemia/lymphoma as well as tropical spastic paraparesis/HTLV-1-associated myelopathy. HTLV-1 is transmitted to T cells through the virological synapse and by extracellular viral assemblies. Here, we uncovered an additional mechanism of virus transmission that is regulated by the HTLV-1-encoded p8 protein. We found that the p8 protein, known to anergize T cells, is also able to increase T-cell contact through lymphocyte function-associated antigen-1 clustering. In addition, p8 augments the number and length of cellular conduits among T cells and is transferred to neighboring T cells through these conduits. p8, by establishing a T-cell network, enhances the envelope-dependent transmission of HTLV-1. Thus, the ability of p8 to simultaneously anergize and cluster T cells, together with its induction of cellular conduits, secures virus propagation while avoiding the hosts immune surveillance. This work identifies p8 as a viral target for the development of therapeutic strategies that may limit the expansion of infected cells in HTLV-1 carriers and decrease HTLV-1-associated morbidity.
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Phase III HIV vaccine trial in Thailand: a step toward a protective vaccine for HIV.
Expert Rev Vaccines
PUBLISHED: 09-09-2010
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The large human efficacy trail in Thailand, RV144, was concluded in the summer of 2009. This is the first Phase III trial to show limited, but significant, efficacy in preventing HIV acquisition. This trial represents the first sign that a preventive vaccine for HIV may be feasible. The vaccine regimen tested in Thailand consisted of priming with a Canarypox vector carrying three synthetic HIV genes. The priming was followed by booster inoculations with two recombinant envelope proteins from HIV, clade B and E. The need to understand the role in protection from HIV acquisition of the new responses, induced by this vaccine combination, has brought together many researchers with the common goal of improving the development of a safe and effective vaccine for HIV.
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Memory T cells in Rhesus macaques.
Adv. Exp. Med. Biol.
PUBLISHED: 08-28-2010
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The Rhesus macaque (Macaca mulatta) is one of the best studied species of Old World monkeys. DNA sequencing of the entire Rhesus macaque genome, completed in 2007, has demonstrated that humans and macaques share about 93% of their nucleotide sequence. Rhesus macaques have been widely used for medical research including drug testing, neurology, behavioral and cognitive science, reproduction, xenotransplantation and genetics. Because of the Rhesus macaques sensitivity to bacteria, parasites and viruses that cause similar disease in humans, these animals represent an excellent model to study infectious diseases. The recent pandemic of HIV and the discovery of SIV, a lentivirus genetically related to HIV Type 1 that causes AIDS in Rhesus macaques, have prompted the development of reagents that can be used to study innate and adaptive immune responses in macaques at the single cell level. This review will focus on the distribution of memory cells in the different immunologic compartments of Rhesus macaques. In addition, the strategies available to manipulate memory cells in Rhesus macaques to understand their trafficking and function will be discussed. Emphasis is placed on studies of memory cells in macaques infected with SIV because many studies are available. Lastly, we highlight the usefulness of the Rhesus macaque model in studies related to the aging of the immune system.
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Requirement of the human T-cell leukemia virus p12 and p30 products for infectivity of human dendritic cells and macaques but not rabbits.
Blood
PUBLISHED: 07-20-2010
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The identification of the genes necessary for human T-cell leukemia virus (HTLV-1) persistence in humans may provide targets for therapeutic approaches. We demonstrate that ablation of the HTLV-1 genes encoding p12, p30, or the HBZ protein, does not affect viral infectivity in rabbits and in this species, only the absence of HBZ is associated with a consistent reduction in virus levels. We observed reversion of the HTLV-1 mutants to the HTLV-1 wild-type genotype in none of the inoculated rabbits. In contrast, in macaques, the absence of HBZ was associated with reversion of the mutant virus to the wild-type genotype in 3 of the 4 animals within weeks from infection. Similarly, reversion to the wild type was observed in 2 of the 4 macaque inoculated with the p30 mutant. The 4 macaques exposed to the p12 knock remained seronegative, and only 2 animals were positive at a single time point for viral DNA in tissues. Interestingly, we found that the p12 and the p30 mutants were also severely impaired in their ability to replicate in human dendritic cells. These data suggest that infection of dendritic cells may be required for the establishment and maintenance of HTLV-1 infection in primate species.
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Hijacking the T-cell communication network by the human T-cell leukemia/lymphoma virus type 1 (HTLV-1) p12 and p8 proteins.
Mol. Aspects Med.
PUBLISHED: 06-23-2010
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The non-structural proteins encoded by the orf-I, II, III, and IV genes of the human T-cell leukemia/lymphoma virus type 1 (HTLV-1) genome, are critical for the modulation of cellular gene expression and T-cell proliferation, the escape from cytotoxic T-cells and natural killer cells, and virus expression. In here, we review the main functions of the HTLV-1 orf-I products. The 12kDa product from orf-I (p12) is proteolytically cleaved within the endoplasmic reticulum (ER) to generate the 8kDa protein (p8). At the steady state, both proteins are expressed at similar levels in transfected T-cells. The p12 protein remains in the ER and cis-Golgi, whereas the p8 protein traffics to the cell surface and is recruited to the immunological synapse. The p12 and the p8 proteins have seemingly opposite effects on T-cells; the ER resident p12, modulates T-cell activation and proliferation, whereas p8 induces T-cell anergy. The p8 protein also increases the formation of cellular conduits, is transferred to neighboring T-cells, and increases virus transmission. The requirement for HTLV-1 infectivity of orf-I is demonstrated by the loss of virus infectivity in macaques exposed to an engineered virus, whereby expression of orf-I was ablated. Altogether the current knowledge demonstrates that the concerted activity of p8 and p12 is essential for the persistence of virus infected cells in the host.
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Th17 cells in pathogenic simian immunodeficiency virus infection of macaques.
Curr Opin HIV AIDS
PUBLISHED: 06-15-2010
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We discuss studies on a subset of CD4T cells, designated Th17, and their role in the pathogenesis of human and simian acquired immune deficiency, caused by infection with HIV and simian immunodeficiency virus (SIV), respectively. Most of the Th17 cells are lost within 2 weeks from infection at mucosal sites of SIV-infected macaques and are not replenished over time. Comparison of simian pathogenic and nonpathogenic models of SIV infection suggests that Th17 cells contribute to the pathogenesis of AIDS.
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Innate and adaptive immune correlates of vaccine and adjuvant-induced control of mucosal transmission of SIV in macaques.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-10-2010
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Adjuvant effects on innate as well as adaptive immunity may be critical for inducing protection against mucosal HIV and simian immunodeficiency virus (SIV) exposure. We therefore studied effects of Toll-like receptor agonists and IL-15 as mucosal adjuvants on both innate and adaptive immunity in a peptide/poxvirus HIV/SIV mucosal vaccine in macaques, and made three critical observations regarding both innate and adaptive correlates of protection: (i) adjuvant-alone without vaccine antigen impacted the intrarectal SIVmac251 challenge outcome, correlating with surprisingly long-lived APOBEC3G (A3G)-mediated innate immunity; in addition, even among animals receiving vaccine with adjuvants, viral load correlated inversely with A3G levels; (ii) a surprising threshold-like effect existed for vaccine-induced adaptive immunity control of viral load, and only antigen-specific polyfunctional CD8(+) T cells correlated with protection, not tetramer(+) T cells, demonstrating the importance of T-cell quality; (iii) synergy was observed between Toll-like receptor agonists and IL-15 for driving adaptive responses through the up-regulation of IL-15Ralpha, which can present IL-15 in trans, as well as for driving the innate A3G response. Thus, strategic use of molecular adjuvants can provide better mucosal protection through induction of both innate and adaptive immunity.
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HTLV-1 p13, a small protein with a busy agenda.
Mol. Aspects Med.
PUBLISHED: 02-11-2010
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Human T-cell leukemia virus type 1 (HTLV-1) infection is characterized by life-long persistence of the virus in the host. While most infected individuals remain asymptomatic, 3-5% will eventually develop adult T-cell leukemia/lymphoma (ATLL) or tropical spastic paraparesis/HTLV-associated myelopathy (TSP/HAM) after a clinical latency that can span years (TSP/HAM) to decades (ATLL). The major oncogenic determinant among HTLV-1 proteins is the Tax transactivator, which influences the expression and function of a great number of cellular proteins, drives cell proliferation, reduces cell death, and induces genetic instability. The present review is focused on the current knowledge of p13, an HTLV-1 accessory protein targeted to the inner mitochondrial membrane and, under certain conditions, to the nucleus. In mitochondria, p13 produces an inward K+current that results in an increased production of ROS by mitochondria. These effects are linked to the proteins effects on cell turnover which include activation of primary T-cells and reduced proliferation/sensitization to death of tumor cells. Recent findings suggest that in the presence of Tax, p13 is subjected to ubiquitylation and partly targeted to the nucleus. Nuclear p13 binds Tax and inhibits its transcriptional activity. These findings suggest that the protein might exert distinct functions depending on its intracellular localization and influence both the turnover of infected cells and the balance between viral latency and productive infection.
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Uncovering the interplay between CD8, CD4 and antibody responses to complex pathogens.
Future Microbiol
PUBLISHED: 02-11-2010
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Vaccinia virus (VACV) was used as the vaccine strain to eradicate smallpox. VACV is still administered to healthcare workers or researchers who are at risk of contracting the virus, and to military personnel. Thus, VACV represents a weapon against outbreaks, both natural (e.g., monkeypox) or man-made (bioterror). This virus is also used as a vector for experimental vaccine development (cancer/infectious disease). As a prototypic poxvirus, VACV is a model system for studying host-pathogen interactions. Until recently, little was known about the targets of host immune responses, which was likely owing to VACVs large genome (>200 open reading frames). However, the last few years have witnessed an explosion of data, and VACV has quickly become a useful model to study adaptive immune responses. This review summarizes and highlights key findings based on identification of VACV antigens targeted by the immune system (CD4, CD8 and antibodies) and the complex interplay between responses.
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Preexisting infection with human T-cell lymphotropic virus type 2 neither exacerbates nor attenuates simian immunodeficiency virus SIVmac251 infection in macaques.
J. Virol.
PUBLISHED: 01-13-2010
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Coinfection with human T-cell lymphotropic virus type 2 (HTLV-2) and human immunodeficiency virus type 1 (HIV-1) has been reported to have either a slowed disease course or to have no effect on progression to AIDS. In this study, we generated a coinfection animal model and investigated whether HTLV-2 could persistently infect macaques, induce a T-cell response, and impact simian immunodeficiency virus SIV(mac251)-induced disease. We found that inoculation of irradiated HTLV-2-infected T cells into Indian rhesus macaques elicited humoral and T-cell responses to HTLV-2 antigens at both systemic and mucosal sites. Low levels of HTLV-2 provirus DNA were detected in the blood, lymphoid tissues, and gastrointestinal tracts of infected animals. Exposure of HTLV-2-infected or naïve macaques to SIV(mac251) demonstrated comparable levels of SIV(mac251) viral replication, similar rates of mucosal and peripheral CD4(+) T-cell loss, and increased T-cell proliferation. Additionally, neither the magnitude nor the functional capacity of the SIV-specific T-cell-mediated immune response was different in HTLV-2/SIV(mac251) coinfected animals versus SIV(mac251) singly infected controls. Thus, HTLV-2 targets mucosal sites, persists, and importantly does not exacerbate SIV(mac251) infection. These data provide the impetus for the development of an attenuated HTLV-2-based vectored vaccine for HIV-1; this approach could elicit persistent mucosal immunity that may prevent HIV-1/SIV(mac251) infection.
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Antiretroviral activity of the aminothiol WR1065 against Human Immunodeficiency virus (HIV-1) in vitro and Simian Immunodeficiency virus (SIV) ex vivo.
AIDS Res Ther
PUBLISHED: 10-15-2009
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WR1065 is the free-thiol metabolite of the cytoprotective aminothiol amifostine, which is used clinically at very high doses to protect patients against toxicity induced by radiation and chemotherapy. In an earlier study we briefly reported that the aminothiol WR1065 also inhibits HIV-1 replication in phytohemagglutinin (PHA)-stimulated human T-cell blasts (TCBs) infected in culture for 2 hr before WR1065 exposure. In this study we expanded the original observations to define the dose-response curve for that inhibition, and address the question of additive effects for the combination of WR1065 plus Zidovudine (AZT). Here we also explored the effect of WR1065 on SIV by examining TCBs taken from macaques with well-established infections several months with SIV.
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Human regulatory T cells are targets for human immunodeficiency Virus (HIV) infection, and their susceptibility differs depending on the HIV type 1 strain.
J. Virol.
PUBLISHED: 10-14-2009
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Regulatory T cells (Treg) are a subpopulation of CD4(+) T cells characterized by the suppressive activity they exert on effector immune responses, including human immunodeficiency virus (HIV)-specific immune responses. Because Treg express CXCR4 and CCR5, they represent potential targets for HIV; however, Treg susceptibility to HIV infection is still unclear. We therefore performed an extensive study of Treg susceptibility to HIV, using lab strains and primary isolates with either CCR5 or CXCR4 tropism. Furthermore, we quantified HIV infection at early and late time points of the virus life cycle. We found that Treg were clearly susceptible to HIV infection. Circulating Treg were not preferentially infected with HIV compared to effector T cells (Teff) in vivo. Conversely, in vitro infection with either CCR5-using (R5) or CXCR4-using (X4) viruses occurred with different dynamics. For instance, HIV infection by R5 viruses (lab strains and primary isolates) resulted in lower levels of infection in Treg compared with Teff at both early and late time points. In contrast, X4 viruses induced higher levels of infection in Treg compared to Teff at early time points, but this difference disappeared at the late time points of the virus life cycle. Our results suggest that the relative susceptibility of Treg to HIV infection compared to Teff varies, depending on both viral and host factors. These variations may play an important role in HIV pathogenesis.
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In vivo genetic mutations define predominant functions of the human T-cell leukemia/lymphoma virus p12I protein.
Blood
PUBLISHED: 05-16-2009
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The human T-cell leukemia/lymphoma virus type 1 (HTLV-1) ORF-I encodes a 99-amino acid hydrophobic membrane protein, p12(I), that affects receptors in different cellular compartments. We report here that proteolytic cleavage dictates different cellular localization and functions of p12(I). The removal of a noncanonical endoplasmic reticulum (ER) retention/retrieval signal within the amino terminus of p12(I) is necessary for trafficking to the Golgi apparatus and generation of a completely cleaved 8-kDa protein. The 8-kDa protein in turn traffics to the cell surface, is recruited to the immunologic synapse following T-cell receptor (TCR) ligation, and down-regulates TCR proximal signaling. The uncleaved 12-kDa form of p12(I) resides in the ER and interacts with the beta and gamma(c) chains of the interleukin-2 receptor (IL-2R), the heavy chain of the major histocompatibility complex (MHC) class I, as well as calreticulin and calnexin. Genetic analysis of ORF-I from ex vivo samples of HTLV-1-infected patients reveals predominant amino acid substitutions within ORF-I that affect proteolytic cleavage, suggesting that ER-associated functions of p12(I) may contribute to the survival and proliferation of the infected T cells in the host.
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Combined effect of antiretroviral therapy and blockade of IDO in SIV-infected rhesus macaques.
J. Immunol.
PUBLISHED: 03-21-2009
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Increased activity of IDO, which catalyzes the degradation of Trp into kynurenine (Kyn), is observed during HIV/SIV infection, and it may contribute to the persistence of HIV/SIV by suppressing antiviral T cell responses. We administered the IDO inhibitor 1-methyl-d-tryptophan (d-1mT) for 13 days to SIV-infected rhesus macaques receiving antiretroviral therapy (ART). d-1mT treatment increased the plasma levels of Trp, without reducing the levels of Kyn, suggesting only a partial effect on IDO enzymatic activity. Surprisingly, d-1mT significantly reduced the virus levels in plasma and lymph nodes of ART-treated animals with incomplete responsiveness to ART. In SIV-infected animals that were not receiving ART, d-1mT was ineffective in reducing the plasma viral load and had only a marginal effect on the plasma Kyn/Trp ratio. Increased IDO and TGF-beta mRNA expression in lymph nodes of ART-treated macaques after d-1mT treatment suggested that compensatory counterregulatory mechanisms were activated by d-1mT, which may account for the lack of effect on plasma Kyn. Finally, d-1mT did not interfere with the ART-induced T cell dynamics in lymph nodes (increased frequency of total CD4 T cells, increase of CD8 T cells expressing the antiapoptotic molecule Bcl2, and reduction of regulatory T cells). Thus, d-1mT appeared to synergize with ART in inhibiting viral replication and did not interfere with the beneficial immunologic effects of ART. Further studies are required to elucidate the immunologic or virologic mechanism by which d-1mT inhibited SIV replication in vivo.
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HAART reduces death ligand but not death receptors in lymphoid tissue of HIV-infected patients and simian immunodeficiency virus-infected macaques.
AIDS
PUBLISHED: 02-21-2009
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To determine how antiretroviral therapy (ART) or HAART affects the expression of apoptotic ligands and their death receptors in the blood and lymphoid tissues of HIV-infected patients and simian immunodeficiency virus-infected macaques.
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Antibodies with high avidity to the gp120 envelope protein in protection from simian immunodeficiency virus SIV(mac251) acquisition in an immunization regimen that mimics the RV-144 Thai trial.
J. Virol.
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The recombinant canarypox vector, ALVAC-HIV, together with human immunodeficiency virus (HIV) gp120 envelope glycoprotein, has protected 31.2% of Thai individuals from HIV acquisition in the RV144 HIV vaccine trial. This outcome was unexpected, given the limited ability of the vaccine components to induce CD8(+) T-cell responses or broadly neutralizing antibodies. We vaccinated macaques with an immunization regimen intended to mimic the RV144 trial and exposed them intrarectally to a dose of the simian immunodeficiency virus SIV(mac251) that transmits few virus variants, similar to HIV transmission to humans. Vaccination induced anti-envelope antibodies in all vaccinees and CD4(+) and CD8(+) T-cell responses. Three of the 11 macaques vaccinated with ALVAC-SIV/gp120 were protected from SIV(mac251) acquisition, but the result was not significant. The remaining vaccinees were infected and progressed to disease. The magnitudes of vaccine-induced SIV(mac251)-specific T-cell responses and binding antibodies were not significantly different between protected and infected animals. However, sera from protected animals had higher avidity antibodies to gp120, recognized the variable envelope regions V1/V2, and reduced SIV(mac251) infectivity in cells that express high levels of ?(4)?(7) integrins, suggesting a functional role of antibodies to V2. The current results emphasize the utility of determining the titer of repeated mucosal challenge in the preclinical evaluation of HIV vaccines.
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Preclinical evaluation of HIV eradication strategies in the simian immunodeficiency virus-infected rhesus macaque: a pilot study testing inhibition of indoleamine 2,3-dioxygenase.
AIDS Res. Hum. Retroviruses
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Even in the setting of maximally suppressive antiretroviral therapy (ART), HIV persists indefinitely. Several mechanisms might contribute to this persistence, including chronic inflammation and immune dysfunction. In this study, we have explored a preclinical model for the evaluation of potential interventions that might serve to eradicate or to minimize the level of persistent virus. Given data that metabolic products of the inducible enzyme indoleamine 2,3-dioxygeanse (IDO) might foster inflammation and viral persistence, chronically simian immunodeficiency virus (SIV)-infected, ART-treated rhesus macaques were treated with the IDO inhibitor 1-methyl tryptophan (1mT). Orally administered 1mT achieved targeted plasma levels, but did not impact tryptophan metabolism or decrease viral RNA or DNA in plasma or in intestinal tissues beyond levels achieved by ART alone. Animals treated with 1mT showed no difference in the levels of T cell activation or differentiation, or in the kinetics or magnitude of viral rebound following cessation of ART. Notwithstanding these negative results, our observations suggest that the chronically SIV-infected rhesus macaque on suppressive ART can serve as a tractable model in which to test and to prioritize the selection of other potential interventions designed to eradicate HIV in vivo. In addition, this model might be used to optimize the route and dose by which such interventions are administered and the methods by which their effects are monitored.
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