Human Macrophages Exhibit GM-CSF Dependent Restriction of Mycobacterium tuberculosis Infection via Regulating Their Self-Survival, Differentiation and Metabolism

GM-CSF is an important cytokine that regulates the proliferation of monocytes/macrophages and its various functions during health and disease. Although growing evidences support the notion that GM-CSF could play a major role in immunity against tuberculosis (TB) infection, the mechanism of GM-CSF mediated protective effect against TB remains largely unknown. Here in this study we examined the secreted levels of GM-CSF by human macrophages from different donors along with the GM-CSF dependent cellular processes that are critical for control of M. tuberculosis infection. While macrophage of different donors varied in their ability to produce GM-CSF, a significant correlation was observed between secreted levels of GM-CSF, survial of macrophages and intra-macrophage control of Mycobacterium tuberculosis bacilli. GM-CSF levels secreted by macrophages negatively correlated with the intra-macrophage M. tuberculosis burden, survival of infected host macrophages positively correlated with their GM-CSF levels. GM-CSF-dependent prolonged survival of human macrophages also correlated with significantly decreased bacterial burden and increased expression of self-renewal/cell-survival associated genes such as BCL-2 and HSP27. Antibody-mediated depletion of GM-CSF in macrophages resulted in induction of significantly elevated levels of apoptotic/necrotic cell death and a simultaneous decrease in autophagic flux. Additionally, protective macrophages against M. tuberculosis that produced more GM-CSF, induced a stronger granulomatous response and produced significantly increased levels of IL-1β, IL-12 and IL-10 and decreased levels of TNF-α and IL-6. In parallel, macrophages isolated from the peripheral blood of active TB patients exhibited reduced capacity to control the intracellular growth of M. tuberculosis and produced significantly lower levels of GM-CSF. Remarkably, as compared to healthy controls, macrophages of active TB patients exhibited significantly altered metabolic state correlating with their GM-CSF secretion levels. Altogether, these results suggest that relative levels of GM-CSF produced by human macrophages plays a critical role in preventing cell death and maintaining a protective differentiation and metabolic state of the host cell against M. tuberculosis infection

Immunization with Single-Cycle SIV Significantly Reduces Viral Loads After an Intravenous Challenge with SIVmac239

Strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection were evaluated for the ability to elicit protective immunity against wild-type SIVmac239 infection of rhesus macaques by two different vaccine regimens. Six animals were inoculated at 8-week intervals with 6 identical doses consisting of a mixture of three different envelope variants of single-cycle SIV (scSIV). Six additional animals were primed with a mixture of cytoplasmic domain-truncated envelope variants of scSIV and boosted with two doses of vesicular stomatitis virus glycoprotein (VSV G) trans-complemented scSIV. While both regimens elicited detectable virus-specific T cell responses, SIV-specific T cell frequencies were more than 10-fold higher after boosting with VSV G trans-complemented scSIV (VSV G scSIV). Broad T cell recognition of multiple viral antigens and Gag-specific CD4+ T cell responses were also observed after boosting with VSV G scSIV. With the exception of a single animal in the repeated immunization group, all of the animals became infected following an intravenous challenge with SIVmac239. However, significantly lower viral loads and higher memory CD4+ T cell counts were observed in both immunized groups relative to an unvaccinated control group. Indeed, both scSIV immunization regimens resulted in containment of SIVmac239 replication after challenge that was as good as, if not better than, what has been achieved by other non-persisting vaccine vectors that have been evaluated in this challenge model. Nevertheless, the extent of protection afforded by scSIV was not as good as typically conferred by persistent infection with live, attenuated SIV. These observations have potentially important implications to the design of an effective AIDS vaccine, since they suggest that ongoing stimulation of virus-specific immune responses may be essential to achieving the degree of protection afforded by live, attenuated SIV

Dramatic Rise in Plasma Viremia after CD8+ T Cell Depletion in Simian Immunodeficiency Virus–infected Macaques

To determine the role of CD8+ T cells in controlling simian immunodeficiency virus (SIV) replication in vivo, we examined the effect of depleting this cell population using an anti-CD8 monoclonal antibody, OKT8F. There was on average a 99.9% reduction of CD8 cells in peripheral blood in six infected Macaca mulatta treated with OKT8F. The apparent CD8 depletion started 1 h after antibody administration, and low CD8 levels were maintained until day 8. An increase in plasma viremia of one to three orders of magnitude was observed in five of the six macaques. The injection of a control antibody to an infected macaque did not induce a sustained viral load increase, nor did it significantly reduce the number of CD8+ T cells. These results demonstrate that CD8 cells play a crucial role in suppressing SIV replication in vivo

Harmonization of the intracellular cytokine staining assay

Active immunotherapy for cancer is an accepted treatment modality aiming to reinforce the T-cell response to cancer. T-cell reactivity is measured by various assays and used to guide the clinical development of immunotherapeutics. However, data obtained across different institutions may vary substantially making comparative conclusions difficult. The Cancer Immunotherapy Immunoguiding Program organizes proficiency panels to identify key parameters influencing the outcome of commonly used T-cell assays followed by harmonization. Our successes with IFNγ-ELISPOT and peptide HLA multimer analysis have led to the current study on intracellular cytokine staining (ICS). We report the results of three successive panels evaluating this assay. At the beginning, 3 out of 9 participants (33 %) were able to detect >6 out of 8 known virus-specific T-cell responses in peripheral blood of healthy individuals. This increased to 50 % of the laboratories in the second phase. The reported percentages of cytokine-producing T cells by the different laboratories were highly variable with coefficients of variation well over 60 %. Variability could partially be explained by protocol-related differences in background cytokine production leading to sub-optimal signal-to-noise ratios. The large number of protocol variables prohibited identification of prime guidelines to harmonize the assays. In addition, the gating strategy used to identify reactive T cells had a major impact on assay outcome. Subsequent harmonization of the gating strategy considerably reduced the variability within the group of participants. In conclusion, we propose that first basic guidelines should be applied for gating in ICS experiments before harmonizing assay protocol variables

A Decline in CCL3-5 Chemokine Gene Expression during Primary Simian-Human Immunodeficiency Virus Infection

BACKGROUND: The CC-chemokines CCL3, CCL4 and CCL5 have been found to block the entry of CCR5-tropic HIV into host cells and to suppress the viral replication in vitro, but the in vivo role of endogenous CC-chemokines in HIV-1 infection is still incompletely understood. METHODOLOGY/PRINCIPLE FINDINGS: In this study, the primate host CCL3, CCL4 and CCL5 gene expression was evaluated in response to simian-human immunodeficiency virus (SHIV) infection in rhesus macaque model. Five rhesus macaques were inoculated with CCR5-tropic SHIV(SF162P4). The mRNA levels of CCL3, CCL4 and CCL5 were measured by real-time PCR at post inoculation day (PID) 0, 7, 14, 21, 35, 56 and 180 in peripheral blood. In addition, a selected subset of samples from CXCR4-tropic SHIV(Ku1)-infected macaques was included with objective to compare the differences in CC-chemokine down-regulation caused by the two SHIVs. Gut-associated lymphoid tissues (GALT) collected from SHIV(SF162P4)-infected animals were also tested by flow cytometry and confocal microscopy to corroborate the gene expression results. Predictably, higher viral loads and CD4+ T cell losses were observed at PID 14 in macaques infected with SHIV(Ku1) than with SHIV(SF162P4). A decline in CC-chemokine gene expression was also found during primary (PID 7-21), but not chronic (PID 180) stage of infection. CONCLUSIONS: It was determined that A) SHIV(SF162P4) down-regulated the CC-chemokine gene expression during acute stage of infection to a greater extent (p<0.05) than SHIV(Ku1), and B) such down-regulation was not paralleled with the CD4+ T cell depletion. Evaluation of CC-chemokine enhancing immunomodulators such as synthetic CpG-oligonucleotides could be explored in future HIV vaccine studies

Vaccine protection against simian immunodeficiency virus in monkeys using recombinant gamma-2 herpesvirus

Recombinant strains of replication-competent rhesus monkey rhadinovirus (RRV) were constructed in which strong promoter/enhancer elements were used to drive expression of simian immunodeficiency virus (SIV) Env or Gag or a Rev-Tat-Nef fusion protein. Cultured rhesus monkey fibroblasts infected with each recombinant strain were shown to express the expected protein. Three RRV-negative and two RRV-positive rhesus monkeys were inoculated intravenously with a mixture of these three recombinant RRVs. Expression of SIV Gag was readily detected in lymph node biopsy specimens taken at 3 weeks postimmunization. Impressive anti-SIV cellular immune responses were elicited on the basis of major histocompatibility complex (MHC) tetramer staining and gamma interferon enzyme-linked immunospot (ELISPOT) assays. Responses were much greater in magnitude in the monkeys that were initially RRV negative but were still readily detected in the two monkeys that were naturally infected with RRV at the time of immunization. By 3 weeks postimmunization, responses measured by MHC tetramer staining in the two Mamu-A*01(+) RRV-negative monkeys reached 9.3% and 13.1% of all CD8(+) T cells in peripheral blood to the Gag CM9 epitope and 2.3% and 7.3% of all CD8(+) T cells in peripheral blood to the Tat SL8 epitope. Virus-specific CD8(+) T cell responses persisted at high levels up to the time of challenge at 18 weeks postimmunization, and responding cells maintained an effector memory phenotype. Despite the ability of the RRVenv recombinant to express high levels of Env in cultured cells, and despite the appearance of strong anti-RRV antibody responses in immunized monkeys, anti-Env antibody responses were below our ability to detect them. Immunized monkeys, together with three unimmunized controls, were challenged intravenously with 10 monkey infectious doses of SIVmac239. All five immunized monkeys and all three controls became infected with SIV, but peak viral loads were 1.2 to 3.0 log(10) units lower and chronic-phase viral loads were 1.0 to 3.0 log(10) units lower in immunized animals than the geometric mean of unimmunized controls. These differences were statistically significant. Anti-Env antibody responses following challenge indicated an anamnestic response in the vaccinated monkeys. These findings further demonstrate the potential of recombinant herpesviruses as preventive vaccines for AIDS. We hypothesize that this live, replication-competent, persistent herpesvirus vector could match, or come close to matching, live attenuated strains of SIV in the degree of protection if the difficulty with elicitation of anti-Env antibody responses can be overcome

The Duration of Antigen-Stimulation Significantly Alters the Diversity of Multifunctional CD4 T Cells Measured by Intracellular Cytokine Staining

The assessment of antigen-specific T cell responses by intracellular cytokine staining (ICS) has become a routine technique in studies of vaccination and immunity. Here, we highlight how the duration of in vitro antigen pre-stimulation, combined with the cytokine accumulation period, are critical parameters of these methods. The effect of varying these parameters upon the diversity and frequency of multifunctional CD4 T cell subsets has been investigated using a murine model of TB vaccination and in cattle naturally infected with Mycobacterium bovis. We demonstrate a substantial influence of the duration of the antigen pre-stimulation period on the repertoire of the antigen-specific CD4 T cell responses. Increasing pre-stimulation from 2 to 6 hours amplified the diversity of the seven potential multifunctional CD4 T cell subsets that secreted any combination of IFN-γ, IL-2 and TNF-α. However, increasing pre-stimulation from 6 to 16 hours markedly altered the multifunctional CD4 T cell repertoire to a dominant IFN-γ+ only response. This was observed in both murine and cattle models

Different Pattern of Immunoglobulin Gene Usage by HIV-1 Compared to Non-HIV-1 Antibodies Derived from the Same Infected Subject

A biased usage of immunoglobulin (Ig) genes is observed in human anti-HIV-1 monoclonal antibodies (mAbs) resulting probably from compensation to reduced usage of the VH3 family genes, while the other alternative suggests that this bias usage is due to antigen requirements. If the antigen structure is responsible for the preferential usage of particular Ig genes, it may have certain implications for HIV vaccine development by the targeting of particular Ig gene-encoded B cell receptors to induce neutralizing anti-HIV-1 antibodies. To address this issue, we have produced HIV-1 specific and non-HIV-1 mAbs from an infected individual and analyzed the Ig gene usage. Green-fluorescence labeled virus-like particles (VLP) expressing HIV-1 envelope (Env) proteins of JRFL and BaL and control VLPs (without Env) were used to select single B cells for the production of 68 recombinant mAbs. Ten of these mAbs were HIV-1 Env specific with neutralizing activity against V3 and the CD4 binding site, as well as non-neutralizing mAbs to gp41. The remaining 58 mAbs were non-HIV-1 Env mAbs with undefined specificities. Analysis revealed that biased usage of Ig genes was restricted only to anti-HIV-1 but not to non-HIV-1 mAbs. The VH1 family genes were dominantly used, followed by VH3, VH4, and VH5 among anti-HIV-1 mAbs, while non-HIV-1 specific mAbs preferentially used VH3 family genes, followed by VH4, VH1 and VH5 families in a pattern identical to Abs derived from healthy individuals. This observation suggests that the biased usage of Ig genes by anti-HIV-1 mAbs is driven by structural requirements of the virus antigens rather than by compensation to any depletion of VH3 B cells due to autoreactive mechanisms, according to the gp120 superantigen hypothesis

Role of complement and antibodies in controlling infection with pathogenic simian immunodeficiency virus (SIV) in macaques vaccinated with replication-deficient viral vectors

<p>Abstract</p> <p>Background</p> <p>We investigated the interplay between complement and antibodies upon priming with single-cycle replicating viral vectors (SCIV) encoding SIV antigens combined with Adeno5-SIV or SCIV pseudotyped with murine leukemia virus envelope boosting strategies. The vaccine was applied via spray-immunization to the tonsils of rhesus macaques and compared with systemic regimens.</p> <p>Results</p> <p>Independent of the application regimen or route, viral loads were significantly reduced after challenge with SIVmac239 (p < 0.03) compared to controls. Considerable amounts of neutralizing antibodies were induced in systemic immunized monkeys. Most of the sera harvested during peak viremia exhibited a trend with an inverse correlation between complement C3-deposition on viral particles and plasma viral load within the different vaccination groups. In contrast, the amount of the observed complement-mediated lysis did not correlate with the reduction of SIV titres.</p> <p>Conclusion</p> <p>The heterologous prime-boost strategy with replication-deficient viral vectors administered exclusively via the tonsils did not induce any neutralizing antibodies before challenge. However, after challenge, comparable SIV-specific humoral immune responses were observed in all vaccinated animals. Immunization with single cycle immunodeficiency viruses mounts humoral immune responses comparable to live-attenuated immunodeficiency virus vaccines.</p