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1.  Three-Dimensional Imaging of HIV-1 Virological Synapses Reveals Membrane Architectures Involved in Virus Transmission 
Journal of Virology  2014;88(18):10327-10339.
HIV transmission efficiency is greatly increased when viruses are transmitted at virological synapses formed between infected and uninfected cells. We have previously shown that virological synapses formed between HIV-pulsed mature dendritic cells (DCs) and uninfected T cells contain interdigitated membrane surfaces, with T cell filopodia extending toward virions sequestered deep inside invaginations formed on the DC membrane. To explore membrane structural changes relevant to HIV transmission across other types of intercellular conjugates, we used a combination of light and focused ion beam scanning electron microscopy (FIB-SEM) to determine the three-dimensional (3D) architectures of contact regions between HIV-1-infected CD4+ T cells and either uninfected human CD4+ T cells or human fetal astrocytes. We present evidence that in each case, membrane extensions that originate from the uninfected cells, either as membrane sheets or filopodial bridges, are present and may be involved in HIV transmission from infected to uninfected cells. We show that individual virions are distributed along the length of astrocyte filopodia, suggesting that virus transfer to the astrocytes is mediated, at least in part, by processes originating from the astrocyte itself. Mechanisms that selectively disrupt the polarization and formation of such membrane extensions could thus represent a possible target for reducing viral spread.
IMPORTANCE Our findings lead to new insights into unique aspects of HIV transmission in the brain and at T cell-T cell synapses, which are thought to be a predominant mode of rapid HIV transmission early in the infection process.
PMCID: PMC4178837  PMID: 24965444
2.  Reduced Inflammation and Lymphoid Tissue Immunopathology in Rhesus Macaques Receiving Anti–Tumor Necrosis Factor Treatment During Primary Simian Immunodeficiency Virus Infection 
The Journal of Infectious Diseases  2012;207(6):880-892.
Background. Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections induce robust, generalized inflammatory responses that begin during acute infection and lead to pathological systemic immune activation, fibrotic damage of lymphoid tissues, and CD4+ T-cell loss, pathogenic processes that contribute to disease progression.
Methods. To better understand the contribution of tumor necrosis factor (TNF), a key regulator of acute inflammation, to lentiviral pathogenesis, rhesus macaques newly infected with SIVmac239 were treated for 12 weeks in a pilot study with adalimumab (Humira), a human anti-TNF monoclonal antibody.
Results. Adalimumab did not affect plasma SIV RNA levels or measures of T-cell immune activation (CD38 or Ki67) in peripheral blood or lymph node T cells. However, compared with untreated rhesus macaques, adalimumab-treated rhesus macaques showed attenuated expression of proinflammatory genes, decreased infiltration of polymorphonuclear cells into the T-cell zone of lymphoid tissues, and weaker antiinflammatory regulatory responses to SIV infection (ie, fewer presumed alternatively activated [ie, CD163+] macrophages, interleukin 10–producing cells, and transforming growth factor β–producing cells), along with reduced lymphoid tissue fibrosis and better preservation of CD4+ T cells.
Conclusions. While HIV/SIV replication drives pathogenesis, these data emphasize the contribution of the inflammatory response to lentiviral infection to overall pathogenesis, and they suggest that early modulation of the inflammatory response may help attenuate disease progression.
PMCID: PMC3571439  PMID: 23087435
SIV; rhesus macaque; Sooty mangabey; lymph node; inflammation; adalimumab; TNF; macrophage; fibrosis; collagen; TGFb
3.  Cytotoxic Capacity of SIV-Specific CD8+ T Cells against Primary Autologous Targets Correlates with Immune Control in SIV-Infected Rhesus Macaques 
PLoS Pathogens  2013;9(2):e1003195.
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.
Author Summary
Clues regarding the features of effective immunity against lentiviruses have come from the study of non-human primates. We evaluated rhesus macaques infected with Simian Immunodeficiency Virus (SIV), a lentivirus closely related to Human Immunodeficiency Virus (HIV). In contrast to most SIV-infected rhesus macaques that develop progressive disease, a small proportion are able to control SIV replication and remain healthy for prolonged durations. In this study, we found that these long-term nonprogressor/elite controller (LTNP/EC) macaques have CD8+ T cells that are extremely effective at killing SIV-infected cells. It seems that this control is mediated by the efficient delivery of active granzyme B, a key molecule involved in the elimination of virus-infected cells. Furthermore, we correctly predicted the presence or absence of control of SIV infection in the majority of animals through measurements of the killing capacity of their CD8+ T cells. These findings indicate that measuring these functions could be used in the evaluation of vaccines against SIV in non-human primates.
PMCID: PMC3585127  PMID: 23468632
4.  Restricted Replication of Xenotropic Murine Leukemia Virus-Related Virus in Pigtailed Macaques 
Journal of Virology  2012;86(6):3152-3166.
Although xenotropic murine leukemia virus-related virus (XMRV) has been previously linked to prostate cancer and myalgic encephalomyelitis/chronic fatigue syndrome, recent data indicate that results interpreted as evidence of human XMRV infection reflect laboratory contamination rather than authentic in vivo infection. Nevertheless, XMRV is a retrovirus of undefined pathogenic potential that is able to replicate in human cells. Here we describe a comprehensive analysis of two male pigtailed macaques (Macaca nemestrina) experimentally infected with XMRV. Following intravenous inoculation with >1010 RNA copy equivalents of XMRV, viral replication was limited and transient, peaking at ≤2,200 viral RNA (vRNA) copies/ml plasma and becoming undetectable by 4 weeks postinfection, though viral DNA (vDNA) in peripheral blood mononuclear cells remained detectable through 119 days of follow-up. Similarly, vRNA was not detectable in lymph nodes by in situ hybridization despite detectable vDNA. Sequencing of cell-associated vDNA revealed extensive G-to-A hypermutation, suggestive of APOBEC-mediated viral restriction. Consistent with limited viral replication, we found transient upregulation of type I interferon responses that returned to baseline by 2 weeks postinfection, no detectable cellular immune responses, and limited or no spread to prostate tissue. Antibody responses, including neutralizing antibodies, however, were detectable by 2 weeks postinfection and maintained throughout the study. Both animals were healthy for the duration of follow-up. These findings indicate that XMRV replication and spread were limited in pigtailed macaques, predominantly by APOBEC-mediated hypermutation. Given that human APOBEC proteins restrict XMRV infection in vitro, human XMRV infection, if it occurred, would be expected to be characterized by similarly limited viral replication and spread.
PMCID: PMC3302341  PMID: 22238316
5.  TCR triggering transcriptionally downregulates CCR5 expression on rhesus macaque CD4+ T cells with no measurable effect on susceptibility to SIV infection 
Virology  2010;409(1):132-140.
Studies using transformed human cell lines suggest that most SIV strains use CCR5 as co-receptor. Our analysis of primary rhesus macaque CD4+ T-cell clones revealed marked differences in susceptibility to SIVmac239 infection. We investigated whether different levels of CCR5 expression account for clonal differences in SIVmac239 susceptibility. Macaque CD4+ T cells showed significant CCR5 downregulation 1-2 days following CD3 mAb stimulation, which gradually recovered at resting state, 7-10 days after activation. Exposure of clones to SIVmac239 during their CCR5low or CCR5high expression states revealed differences in SIV susceptibility independent of surface CCR5 levels. Furthermore, a CCR5 antagonist similarly reduced SIVmac239 infection of clones during their CCR5low or CCR5high expression states. Our data suggest a model where i) very low levels of CCR5 are sufficient for efficient SIV infection, ii) CCR5 levels above this threshold do not enhance infection, and iii) low level infection can occur in the absence of CCR5.
PMCID: PMC3001627  PMID: 21035160
AIDS; CCR5; CD4; SIV; rhesus macaques
6.  Distribution, persistence and efficacy of adoptively transferred central and effector memory-derived autologous SIV-specific CD8+ T-cell clones in rhesus macaques during acute infection1 
Plasma viremia decreases coincident with the appearance of virus-specific CD8+ T cells during acute HIV or SIV infection. This finding, along with demonstrations of viral mutational escape from CD8+ T-cell responses and transient increase in plasma viremia after depletion of CD8+ T cells in SIV-infected monkeys strongly suggest a role for CD8+ T cells in controlling HIV/SIV. However, direct quantitative or qualitative correlates between CD8+ T-cell activity and virus control have not been established. To directly assess the impact of large numbers of virus-specific CD8+ T cells present at time of SIV infection, we transferred in vitro expanded autologous central and effector memory-derived Gag CM9-, Nef YY9- and Vif WY8-specific CD8+ T-cell clones to acutely infected rhesus macaques. The cells persisted in PBMC between 4 and 9 days but were not detected in gut-associated lymphoid tissue or lymph nodes. Interestingly, a high frequency of the infused cells localized to the lungs where they persisted at high frequency for more than 6 weeks. While persisting cells in the lungs were antigen reactive, there was no measurable effect on virus load. Sequencing of virus from the animal receiving Nef YY9-specific CD8+ T cells demonstrated an escape mutation in this epitope less than 3 weeks post infection, consistent with immune selection pressure by the infused cells. These studies establish methods for adoptive transfer of autologous SIV-specific CD8+ T cells for evaluating immune control during acute infection, and demonstrate that infused cells retain function and persist for at least 2 months in specific tissues.
PMCID: PMC2797560  PMID: 19949091
7.  Effector-memory T cell responses are associated with protection of rhesus monkeys from mucosal SIV challenge 
Nature medicine  2009;15(3):293-299.
The rapid onset of massive, systemic viral replication during primary HIV/SIV infection and the immune evasion capabilities of these viruses pose fundamental problems for vaccines that depend upon initial viral replication to stimulate effector T cell expansion and differentiation1–5. We hypothesized that vaccines designed to maintain differentiated “effector memory” T cell (TEM) responses5,6 at viral entry sites might improve efficacy by impairing viral replication at its earliest stage2, and have therefore developed SIV protein-encoding vectors based on rhesus cytomegalovirus (RhCMV), the prototypical inducer of life-long TEM responses7–9. RhCMV vectors expressing SIV Gag, Rev/Nef/Tat, and Env persistently infected rhesus macaques (RM), regardless of pre-existing RhCMV immunity, and primed and maintained robust SIV-specific, CD4+ and CD8+ TEM responses (characterized by coordinate TNF, IFN-γ and MIP-1β expression, cytotoxic degranulation, and accumulation at extra-lymphoid sites) in the absence of neutralizing antibodies. Compared to control RM, these vaccinated RM showed increased resistance to acquisition of progressive SIVmac239 infection upon repeated, limiting dose, intra-rectal challenge, including four animals that controlled rectal mucosal infection without progressive systemic dissemination. These data suggest a new paradigm for AIDS vaccine development: that vaccines capable of generating and maintaining HIV-specific TEM might decrease the incidence of HIV acquisition after sexual exposure.
PMCID: PMC2720091  PMID: 19219024
8.  Proteomic and Biochemical Analysis of Purified Human Immunodeficiency Virus Type 1 Produced from Infected Monocyte-Derived Macrophages 
Journal of Virology  2006;80(18):9039-9052.
Human immunodeficiency virus type 1 (HIV-1) infects CD4+ T lymphocytes and monocytes/macrophages, incorporating host proteins in the process of assembly and budding. Analysis of the host cell proteins incorporated into virions can provide insights into viral biology. We characterized proteins in highly purified HIV-1 virions produced from human monocyte-derived macrophages (MDM), within which virus buds predominantly into intracytoplasmic vesicles, in contrast to the plasmalemmal budding of HIV-1 typically seen with infected T cells. Liquid chromatography-linked tandem mass spectrometry of highly purified virions identified many cellular proteins, including 33 previously described proteins in HIV-1 preparations from other cell types. Proteins involved in many different cellular structures and functions were present, including those from the cytoskeleton, adhesion, signaling, intracellular trafficking, chaperone, metabolic, ubiquitin/proteasomal, and immune response systems. We also identified annexins, annexin-binding proteins, Rab proteins, and other proteins involved in membrane organization, vesicular trafficking, and late endosomal function, as well as apolipoprotein E, which participates in cholesterol transport, immunoregulation, and modulation of cell growth and differentiation. Several tetraspanins, markers of the late endosomal compartment, were also identified. MDM-derived HIV contained 26 of 37 proteins previously found in exosomes, consistent with the idea that HIV uses the late endosome/multivesicular body pathway during virion budding from macrophages.
PMCID: PMC1563931  PMID: 16940516
9.  Development of a cAdVax-Based Bivalent Ebola Virus Vaccine That Induces Immune Responses against both the Sudan and Zaire Species of Ebola Virus 
Journal of Virology  2006;80(6):2738-2746.
Ebola virus (EBOV) causes a severe hemorrhagic fever for which there are currently no vaccines or effective treatments. While lethal human outbreaks have so far been restricted to sub-Saharan Africa, the potential exploitation of EBOV as a biological weapon cannot be ignored. Two species of EBOV, Sudan ebolavirus (SEBOV) and Zaire ebolavirus (ZEBOV), have been responsible for all of the deadly human outbreaks resulting from this virus. Therefore, it is important to develop a vaccine that can prevent infection by both lethal species. Here, we describe the bivalent cAdVaxE(GPs/z) vaccine, which includes the SEBOV glycoprotein (GP) and ZEBOV GP genes together in a single complex adenovirus-based vaccine (cAdVax) vector. Vaccination of mice with the bivalent cAdVaxE(GPs/z) vaccine led to efficient induction of EBOV-specific antibody and cell-mediated immune responses to both species of EBOV. In addition, the cAdVax technology demonstrated induction of a 100% protective immune response in mice, as all vaccinated C57BL/6 and BALB/c mice survived challenge with a lethal dose of ZEBOV (30,000 times the 50% lethal dose). This study demonstrates the potential efficacy of a bivalent EBOV vaccine based on a cAdVax vaccine vector design.
PMCID: PMC1395467  PMID: 16501083
10.  Quantitation of HLA Class II Protein Incorporated into Human Immunodeficiency Type 1 Virions Purified by Anti-CD45 Immunoaffinity Depletion of Microvesicles 
Journal of Virology  2003;77(23):12699-12709.
Among the many host cell-derived proteins found in human immunodeficiency virus type 1 (HIV-1), HLA class II (HLA-II) appears to be selectively incorporated onto virions and may contribute to mechanisms of indirect imunopathogenesis in HIV infection and AIDS. However, the amount of HLA-II on the surface of HIV-1 particles has not been reliably determined due to contamination of virus preparations by microvesicles containing host cell proteins, including HLA-II. Even rigorous sucrose density centrifugation is unable to completely separate HIV-1 from microvesicles. CD45, a leukocyte integral membrane protein, is found on microvesicles, yet appears to be excluded from HIV-1 particles. Exploiting this observation, we have developed a CD45-based immunoaffinity depletion method for removing CD45-containing microvesicles that yields highly purified preparations of virions. Examination of CD45-depleted HIV-1MN by high-pressure liquid chromatography, protein sequencing, and amino acid analyses determined a molar ratio of HLA-II to Gag of 0.04 to 0.05 in the purified virions, corresponding to an estimated average of 50 to 63 native HLA-II complexes (i.e., a dimer of α and β heterodimers) per virion. These values are approximately 5- to 10-fold lower than those previously determined for other virion preparations that contained microvesicles. Our observations demonstrate the utility of CD45 immunoaffinity-based approaches for producing highly purified retrovirus preparations for applications that would benefit from the use of virus that is essentially free of microvesicles.
PMCID: PMC262561  PMID: 14610192
11.  Differential Incorporation of CD45, CD80 (B7-1), CD86 (B7-2), and Major Histocompatibility Complex Class I and II Molecules into Human Immunodeficiency Virus Type 1 Virions and Microvesicles: Implications for Viral Pathogenesis and Immune Regulation 
Journal of Virology  2001;75(13):6173-6182.
Human immunodeficiency virus (HIV) infection results in a functional impairment of CD4+ T cells long before a quantitative decline in circulating CD4+ T cells is evident. The mechanism(s) responsible for this functional unresponsiveness and eventual depletion of CD4+ T cells remains unclear. Both direct effects of cytopathic infection of CD4+ cells and indirect effects in which uninfected “bystander” cells are functionally compromised or killed have been implicated as contributing to the immunopathogenesis of HIV infection. Because T-cell receptor engagement of major histocompatibility complex (MHC) molecules in the absence of costimulation mediated via CD28 binding to CD80 (B7-1) or CD86 (B7-2) can lead to anergy or apoptosis, we determined whether HIV type 1 (HIV-1) virions incorporated MHC class I (MHC-I), MHC-II, CD80, or CD86. Microvesicles produced from matched uninfected cells were also evaluated. HIV infection increased MHC-II expression on T- and B-cell lines, macrophages, and peripheral blood mononclear cells (PBMC) but did not significantly alter the expression of CD80 or CD86. HIV virions derived from all MHC-II-positive cell types incorporated high levels of MHC-II, and both virions and microvesicles preferentially incorporated CD86 compared to CD80. CD45, expressed at high levels on cells, was identified as a protein present at high levels on microvesicles but was not detected on HIV-1 virions. Virion-associated, host cell-derived molecules impacted the ability of noninfectious HIV virions to trigger death in freshly isolated PBMC. These results demonstrate the preferential incorporation or exclusion of host cell proteins by budding HIV-1 virions and suggest that host cell proteins present on HIV-1 virions may contribute to the overall pathogenesis of HIV-1 infection.
PMCID: PMC114333  PMID: 11390619

Results 1-11 (11)