Development and evaluation of a new interferon-gamma release assay for the diagnosis of tuberculosis infection in HIV-infected individuals in China.

BACKGROUND: Human immunodeficiency virus (HIV)-infected individuals are at high risk of contracting tuberculosis (TB) disease, and current methods for diagnosing TB infection are less effective in this population. We developed and evaluated a new interferon-gamma release assay (IGRA), named A.TB, in HIV-infected individuals, with and without active TB, in a setting of high TB burden and low HIV prevalence. METHODS: A total of 255 subjects were divided into 3 groups according to their HIV and TB status: HIV+ without active TB (n = 123), HIV+/TB+ (n = 79), and HIV-/TB+ (n = 65). The A.TB assay was performed in parallel with the QuantiFERON-TB Gold In-Tube (QFT-GIT) and tuberculin skin test (TST). RESULTS: The positive rate was 59.3% (n = 123) by A.TB and 53.8% (n = 106) by QFT-GIT. We observed a strong concordance of 81.2% (k = 0.612) between the two IGRAs. The QFT-GIT results were affected by low CD4(+) cell count (p = 0.013), while A.TB results were not. A.TB was also performed in patients with active TB (n = 65) and patients with active TB and HIV co-infection (n = 79). The sensitivity of A.TB in these groups was 80.0% and 81.0%, respectively. CONCLUSION: The A.TB results were not affected by low CD4(+) cell count in the co-infected cohort. With further evaluation, A.TB may prove to be a valuable tool for diagnosing TB in HIV-infected patients.

High resolution human leukocyte antigen class I allele frequencies and HIV-1 infection associations in Chinese Han and Uyghur cohorts.

BACKGROUND: Host immunogenetic factors such as HLA class I polymorphism are important to HIV-1 infection risk and AIDS progression. Previous studies using high-resolution HLA class I profile data of Chinese populations appeared insufficient to provide information for HIV-1 vaccine development and clinical trial design. Here we reported HLA class I association with HIV-1 susceptibility in a Chinese Han and a Chinese Uyghur cohort. METHODOLOGY/PRINCIPAL FINDINGS: Our cohort included 327 Han and 161 Uyghur ethnic individuals. Each cohort included HIV-1 seropositive and HIV-1 seronegative subjects. Four-digit HLA class I typing was performed by sequencing-based typing and high-resolution PCR-sequence specific primer. We compared the HLA class I allele and inferred haplotype frequencies between HIV-1 seropositive and seronegative groups. A neighbor-joining tree between our cohorts and other populations was constructed based on allele frequencies of HLA-A and HLA-B loci. We identified 58 HLA-A, 75 HLA-B, and 32 HLA-Cw distinct alleles from our cohort and no novel alleles. The frequency of HLA-B*5201 and A*0301 was significantly higher in the Han HIV-1 negative group. The frequency of HLA-B*5101 was significantly higher in the Uyghur HIV-1 negative group. We observed statistically significant increases in expectation-maximization (EM) algorithm predicted haplotype frequencies of HLA-A*0201-B*5101 in the Uyghur HIV-1 negative group, and of Cw*0304-B*4001 in the Han HIV-1 negative group. The B62s supertype frequency was found to be significantly higher in the Han HIV-1 negative group than in the Han HIV-1 positive group. CONCLUSIONS: At the four-digit level, several HLA class I alleles and haplotypes were associated with lower HIV-1 susceptibility. Homogeneity of HLA class I and Bw4/Bw6 heterozygosity were not associated with HIV-1 susceptibility in our cohort. These observations contribute to the Chinese HLA database and could prove useful in the development of HIV-1 vaccine candidates.

Evaluation of a new interferon-gamma release assay and comparison to tuberculin skin test during a tuberculosis outbreak.

BACKGROUND: The tuberculin skin test (TST) is commonly used for the diagnosis of latent tuberculosis infection (LTBI) in non-bacille Calmette-Guérin (BCG) vaccination settings. In recent years, attention has been drawn to interferon-gamma release assays (IGRAs), especially in BCG-vaccinated populations. In this study, we evaluated the TST and a new whole blood IGRA in BCG-vaccinated individuals during a tuberculosis (TB) outbreak in China. METHODS: A TB outbreak occurred at a university in Dalian, China from March to November 2010. The TST and a whole blood IGRA were used to screen for TB infection. The correlation between exposure levels, TST, and the IGRA were evaluated. RESULTS: We found that agreement between the IGRA and TST was poor (kappa 0.182-0.290). IGRA positivity was associated with the level of exposure, and IGRA positivity and the level of exposure were risk factors for TB incidence. Neither the IGRA nor the TST alone picked up all TB incidences. However, if a 10 mm cutoff for the TST was used in the highest risk exposure group and IGRA positivity was used in the other risk groups, 19 of the 20 (95%) TB cases were identified. CONCLUSIONS: A recommended preventive treatment regimen for China should be based on the level of exposure in conjunction with IGRA and TST test results.

HIV vaccine candidates generate in vitro T cell response to putative epitopes in Chinese-origin rhesus macaques.

The Indian rhesus macaque is the established animal model for HIV infection and vaccine research. Growing evidence suggests that the more readily available Chinese rhesus macaque may be a more relevant option. As increasing numbers of novel Chinese rhesus MHC alleles are reported, we decided to explore potential HIV vaccine epitopes in this model. We immunized forty Chinese rhesus macaques with three different HIV vaccine candidates either individually or following a prime/boost strategy. We used ELISPOT to measure immune response in vitro to HIV-1 p24C and HIV-1 gp160 peptide libraries. We identified five putative epitopes with associations to HLA-I alleles including HLA*B-2705 and HLA-B*5101 (associated with slow disease progression and low viral set point) and HLA-B*18 (associated with rapid disease progression and high viral set point). This suggests the possible use of Chinese rhesus macaques to model different disease progressions. We also explored the use of fusion proteins as stimulators in ELISPOT assays. While PBMCs from 6 monkeys responded to peptide stimulation, PBMCs from 28 monkeys responded to the anthrax lethal factor fusion proteins LFn p24C and/or LFn gp140C. Our results support the use of Chinese rhesus macaques in HIV vaccine studies.

Simultaneous detection of antibodies to five simian viruses in nonhuman primates using recombinant viral protein based multiplex microbead immunoassays.

Routine screening for infectious agents is critical in establishing and maintaining specific pathogen free (SPF) nonhuman primate (NHP) colonies. More efficient, higher throughput, less costly reagent, and reduced sample consumption multiplex microbead immunoassays (MMIAs) using purified viral lysates have been developed previously to address some disadvantages of the traditional individual enzyme-linked immunosorbent assay (ELISA) methods. To overcome some of the technical and biosafety difficulties in preparing antigens from live viruses for viral lysate protein based MMIAs, novel MMIAs using recombinant glycoprotein D precursor (gD) protein of herpesvirus B and four viral gag proteins of simian immunodeficiency virus (SIV), simian T Cell lymphotropic virus (STLV), simian foamy virus (SFV), and simian betaretrovirus (SRV) as antigens have been developed in the current study. The data showed that the recombinant viral protein based MMIAs detected simultaneously antibodies to each of these five viruses with high sensitivity and specificity, and correlated well with viral lysate based MMIAs. Therefore, recombinant viral protein based MMIA is an effective and efficient routine screening method to determine the infection status of nonhuman primates.

Recombinant hepatitis B large surface antigen, successfully produced in Escherichia coli, stimulates T-cell response in mice.

A fusion protein consisting of the PA binding domain of anthrax lethal factor (LFn) and a codon optimized Hepatitis B virus large surface antigen (LHBsAg) expresses well in Escherichia coli. The LFn-LHBsAg fusion protein effectively elicits a cell-mediated immune (CMI) response to the hepatitis B viral antigens in mice.

A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen.

Anthrax protective antigen (PA) is a 735-aa polypeptide that facilitates the exit of anthrax lethal factor (LF) from the endosome to the cytosol where the toxin acts. We recently found, however, that a fusion protein of the detoxified N-terminal domain of lethal factor (LFn) with a foreign peptide could induce CD8 T cell immune responses in the absence of PA. Because CD8 T cells recognize peptides derived from proteins degraded in the cytosol, this result suggests that lethal factor may be capable of entering the cytosol independently of PA. To investigate this further, the intracellular trafficking of an LFn-enhanced green fluorescent protein fusion protein (LFn-GFP) in the presence or absence of PA was examined by using confocal microscopy. LFn-GFP is able to enter the cytosol without PA. Moreover, it efficiently colocalizes with the proteosome 20s subunit, which degrades proteins into peptides for presentation to CD8 T cells by the MHC class I pathway. We further demonstrate that in the presence of an immune adjuvant LFn fusion protein without PA is able to effectively elicit anti-HIV cytotoxic T lymphocyte in inbred mice. These results indicate that LFn may be used without PA in a protein vaccine as a carrier to deliver antigens into the cytosol for efficient induction of T lymphocyte responses. Furthermore, these results enable us to propose a modified molecular mechanism of anthrax lethal toxin.

Delivery of exogenous protein antigens to major histocompatibility complex class I pathway in cytosol.

A fragment of anthrax lethal factor possesses the interesting function of delivering recombinant protein antigens through the classical major histocompatibility complex (MHC) class I pathway. This region of the lethal factor lacks the domain associated with anthrax cytotoxicity and functions independently of its binary partner, protective antigen. Experiments that used inhibitors at different steps of the MHC class I pathway supported this hypothesis. Application of this discovery to current T cell assays allows for the measurement of cytotoxic T lymphocyte function without resorting to live vectors and provides a useful new tool to design and test T cell-dependent vaccines.