Development of a highly sensitive liquid chromatography/tandem mass spectrometry method to quantify total and free levels of a target protein, interferon-gamma-inducible protein-10, at picomolar levels in human serum.

RATIONALE: Liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays are increasingly being used for absolute quantitation of proteins due to high specificity and low cost. However, the major challenge for the LC/MS method is insufficient sensitivity. This paper details the strategies developed to maximize the sensitivity from aspects of chromatography, mass spectrometry, and sample preparation to achieve a highly sensitive LC/MS method. METHODS: The method is based on the LC/MS/MS measurement of a surrogate peptide generated from trypsin digestion of interferon-gamma-inducible protein-10 (IP-10). The sample preparation strategy involved selectively extracting IP-10 and removing high-abundance serum proteins through acidified protein precipitation (PPT). It was revealed in this work that these high-abundance serum proteins, if not separated from the protein of interest, could cause significant ionization saturation and high background noise in selected reaction monitoring (SRM), leading to a 100-fold higher lower limit of quantification (LLOQ). RESULTS: Our method demonstrated that the acidified PPT could be optimized to selectively extract the protein of interest with full recovery of 97% to 103%, while the high-abundance serum proteins could be effectively removed with minimal matrix effect of 90% to 93%. For the first time, a highly sensitive LC/MS method with a LLOQ of 31.62 pM for the quantitation of IP-10 has been achieved, which is a 100-fold improvement over the generic method. CONCLUSIONS: The described method offers excellent sensitivity with advantages of being antibody reagent independent and leads to significant cost and time savings. It has been successfully employed to determine both total and free IP-10 levels in human serum samples. This method development strategy may also be applied to other small proteins.

Development of Potential Pharmacodynamic and Diagnostic Markers for Anti-IFN-α Monoclonal Antibody Trials in Systemic Lupus Erythematosus.

To identify potential pharmacodynamic biomarkers to guide dose selection in clinical trials using anti-interferon-alpha (IFN-α) monoclonal antibody (mAb) therapy for systemic lupus erythematosus (SLE), we used an Affymetrix human genome array platform and identified 110 IFN-α/ß-inducible transcripts significantly upregulated in whole blood (WB) of 41 SLE patients. The overexpression of these genes was confirmed prospectively in 54 additional SLE patients and allowed for the categorization of the SLE patients into groups of high, moderate, and weak overexpressers of IFN-α/ß-inducible genes. This approach could potentially allow for an accurate assessment of drug target neutralization in early trials of anti-IFN-α mAb therapy for SLE. Furthermore, ex vivo stimulation of healthy donor peripheral blood mononuclear cells with SLE patient serum and subsequent neutralization with anti-IFN-α mAb or anti-IFN-α receptor mAb showed that anti-IFN-α mAb has comparable effects of neutralizing the overexpression of type I IFN-inducible genes as that of anti-IFNAR mAb. These results suggest that IFN-α, and not other members of type I IFN family in SLE patients, is mainly responsible for the induction of type I IFN-inducible genes in WB of SLE patients. Taken together, these data strengthen the view of IFN-α as a therapeutic target for SLE.

Selenium inhibits 15-hydroperoxyoctadecadienoic acid-induced intracellular adhesion molecule expression in aortic endothelial cells.

Increased intracellular adhesion molecule 1 (ICAM-1) expression and enhanced monocyte recruitment to the endothelium are critical steps in the early development of atherosclerosis. The 15-lipoxygenase 1 (15-LOX1) pathway can generate several proinflammatory eicosanoids that are known to enhance ICAM-1 expression within the vascular endothelium. Oxidative stress can exacerbate endothelial cell inflammatory responses by modifying arachidonic acid metabolism through the 15-LOX1 pathway. Because selenium (Se) influences the oxidant status of cells and can modify the expression of eicosanoids, we investigated the role of this micronutrient in modifying ICAM-1 expression as a consequence of enhanced 15-LOX1 activity. Se supplementation reduced ICAM-1 expression in bovine aortic endothelial cells, an effect that was reversed with 15-LOX1 overexpression or treatment with exogenous 15-hydroperoxyoctadecadienoic acid (15-HPETE). ICAM-1 expression increased proportionately when intracellular15-HPETE levels were allowed to accumulate. However, changes in intracellular 15-HETE levels did not seem to affect ICAM-1 expression regardless of Se status. Our results indicate that Se supplementation can reduce 15-HPETE-induced expression of ICAM-1 by controlling the intracellular accumulation of this fatty acid hydroperoxide in endothelial cells.

Could heme-oxygenase-1 have a role in modulating the recipient immune response to embryonic stem cells?

Pluripotent human embryonic stem cells (hESCs) may provide a potential source of cellular therapies, but as allogeneic cells may require evading the recipient's immune response. Using an NIH-registry hESC line, it was found that undifferentiated hESCs induce a reduced proliferative response compared to PBMC and demonstrate that this diminished response correlates with the activity of heme oxygenase-1 (HO-1). Inhibition of HO-1 significantly increases T cell proliferation against hESC, indicating the potential suppression of these cells during transplantation of allogeneic hESC. These data suggest the hypothesis that HO-1 provides a mechanism for protecting hESCs in vivo.

Evaluation of cellular immune responses in subjects chronically infected with HIV type 1.

The importance of host cellular immune responses, particularly CD8(+) cytotoxic T-lymphocyte (CTL) responses, in control of human immunodeficiency virus type 1 (HIV-1) infection has been demonstrated in many clinical studies. These studies, along with vaccination challenge studies in rhesus macaques, indicate the importance of cellular immune responses against HIV-1. Toward this end, we evaluated anti-HIV-1 cellular immune responses in a cohort of 54 subjects who were chronically infected with HIV-1. By validation of IFN-gamma ELISpot assay, we established a dual cut-off criterion for scoring a positive response. The magnitude and frequency of cellular immune responses were measured against HIV-1 antigens (Gag, Pol, Nef, Rev, and Tat), using synthetic peptides as antigens in ELISpot assay. Here we showed that HIV-1 Gag, Pol, and Nef were frequent targets of T cell responses in these subjects, whereas Tat and Rev were less frequently recognized. We further evaluated the possible association between host cellular immune responses and corresponding plasma viral loads in this cohort. By performing ranking correlation analysis, we demonstrated a positive correlation between host viral loads and ELISpot responses of HIV Gag and Pol in untreated subjects. For the subjects under antiviral regimens, however, we did not find any significant association. Our findings suggest that the high levels of ELISpot responses in chronically infected subjects were reflective of their persistent viral infection.

Thioredoxin reductase regulates the induction of haem oxygenase-1 expression in aortic endothelial cells.

Certain selenoproteins such as GPX-1 (glutathione peroxidase-1) and TrxR1 (thioredoxin reductase-1) possess important antioxidant defence functions in vascular endothelial cells. Reduced selenoprotein activity during dietary selenium (Se) deficiency can result in a compensatory increase of other non-Se-dependent antioxidants, such as HO-1 (haem oxygenase-1) that may help to counteract the damaging effects of oxidant stress. However, the role of individual selenoproteins in regulating vascular-derived protective gene responses such as HO-1 is less understood. Using an oxidant stress model based on Se deficiency in BAECs (bovine aortic endothelial cells), we sought to determine whether TrxR1 activity may contribute to the differential regulation of HO-1 expression as a function of altered redox environment. Se-sufficient BAECs up-regulated HO-1 expression following stimulation with the pro-oxidant, 15-HPETE (15-hydroperoxyeicosatetraenoic acid), and levels of this antioxidant inversely correlated with EC apoptosis. While Se-deficient BAECs exhibited higher basal levels of HO-1, it was not up-regulated upon 15-HPETE treatment, which resulted in significantly higher levels of pro-apoptotic markers. Subsequent results showed that HO-1 induction depended on the activity of TrxR1, as proved with chemical inhibitor studies and direct inhibition with TrxR1 siRNA. Finally, restoring intracellular levels of the reduced substrate Trx (thioredoxin) in Sedeficient BAECs was sufficient to increase HO-1 activation following 15-HPETE stimulation. These data provide evidence for the involvement of the Trx/TrxR system, in the regulation of HO-1 expression in BAECs during pro-oxidant challenge.

Intracellular staining for HIV-specific IFN-gamma production: statistical analyses establish reproducibility and criteria for distinguishing positive responses.

The cellular immune response plays a pivotal role in controlling the spread of HIV-1 infection by lysing virally infected cells and producing potent antiviral cytokines, such as interferon-gamma (IFN-gamma). Flow cytometric methods have been established to evaluate the contribution of both CD4 and CD8 subsets of T lymphocytes to the immune response to HIV by measuring their production of intracellular IFN-gamma following brief antigenic stimulation. We present a statistical treatment of intracellular cytokine staining (ICS) data that is aimed at establishing the reproducibility and robustness of this assay for use in HIV clinical trials. Comparisons of responses from HIV-seronegative and seropositive individuals were used to establish a 2-fold criterion for distinguishing positive responses with a low probability of false positives (<1%). Additional comparisons established that the reproducibility of the assay is between 1.4 and 2.0-fold depending on the magnitude of the response. Little variability was demonstrated between multiple operators for both the execution and analysis components of these experiments (<10% difference with 95% confidence). We conclude that the statistical criteria established by these analyses allow for the accurate detection and comparison of positive responses. Using these statistical criteria, the ICS assay is sufficiently robust for use in HIV-specific vaccine trials.

Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene.

Cellular immune responses, particularly those associated with CD3(+) CD8(+) cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5-gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4(+) and CD8(+) T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5-gag-induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5-gag. The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5-gag. These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.

Mamu-A*01 allele-mediated attenuation of disease progression in simian-human immunodeficiency virus infection.

Expression of several major histocompatibility complex (MHC) class I alleles is associated with a protective effect against disease progression in both human immunodeficiency virus type 1 and simian immunodeficiency virus infection. To understand the mechanism underlying this effect, we investigated the expression of the MHC class I allele Mamu-A*01 in simian-human immunodeficiency virus (SHIV) infection, one of the major models for evaluation of AIDS vaccine candidates. We found that disease progression was significantly delayed in Mamu-A*01-positive rhesus monkeys infected with the highly pathogenic SHIV 89.6P. The delay corresponded not only to a noted Mamu-A*01-restricted dominant cytotoxic T-lymphocyte (CTL) response but also to a lower viral load in lymph nodes (LN) and, importantly, to minimal destruction of LN structure during early infection. In contrast, Mamu-A*01-negative monkeys exhibited massive destruction of LN structure with accompanying rapid disease progression. These data indicate that MHC class I allele-restricted CTL responses may play an important role in preservation of lymphoid tissue structure, thereby resulting in attenuation of disease progression in immunodeficiency virus infection.

Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity.

Recent studies of human immunodeficiency virus type 1 (HIV-1) infection in humans and of simian immunodeficiency virus (SIV) in rhesus monkeys have shown that resolution of the acute viral infection and control of the subsequent persistent infection are mediated by the antiviral cellular immune response. We comparatively assessed several vaccine vector delivery systems-three formulations of a plasmid DNA vector, the modified vaccinia Ankara (MVA) virus, and a replication incompetent adenovirus type 5 (Ad5) vector-expressing the SIV gag protein for their ability to elicit such immune responses in monkeys. The vaccines were tested either as a single modality or in combined modality regimens. Here we show that the most effective responses were elicited by a replication-incompetent Ad5 vector, used either alone or as a booster inoculation after priming with a DNA vector. After challenge with a pathogenic HIV-SIV hybrid virus (SHIV), the animals immunized with Ad5 vector exhibited the most pronounced attenuation of the virus infection. The replication-defective adenovirus is a promising vaccine vector for development of an HIV-1 vaccine.