Synergistic induction of interferon-γ by interleukin-2, interleukin-12 and poly(I:C) in a human natural killer cell line.

Natural killer (NK) cells express various Toll-like receptors (TLRs). Little is known about the role of TLRs in direct NK cell activation. To clarify possible synergistic roles of cytokine and TLR signaling, human NK cell line KHYG-1 was stimulated with agonists for TLR1-9. IFN-γ production was not significantly induced following stimulation with single TLR agonists. Of the nine TLR agonists tested, only poly(I:C) strongly upregulated IFN-γ production by synergistic interleukin-2 (IL-2) and IL-12 stimulation. The role of TLR3 signaling was also examined. An inhibitor of Toll-IL-1 receptor domain-containing adaptor inducing IFN-ß (TRIF) blocked this synergistic action. However, TLR3 expression was unchanged in the presence of IL-2 and IL-12. Our findings suggest a possible role for type 1 cytokines in NK cell IFN-γ production against viral infections.

Seroprevalence of Chlamydophila pneumoniae in HIV-infected children in Vietnam.

A total of 89 blood samples collected from HIV-infected infants and children from provinces of southern Vietnam who were hospitalized at Children's Hospital 1, Ho Chi Minh City, during the 1-year period from October 2004 to September 2005 were submitted to serological screening for IgG, IgA, and IgM antibodies against Chlamydophila pneumoniae (C. pneumoniae). The presence of this microorganism was also evaluated by PCR. The results showed that 64 % of the samples were positive for anti-C. pneumoniae IgG, 31.5 % were positive for IgA, and 3.4 % were positive for IgM. The highest prevalences of IgG and IgA positivity, 75 % and 66.7 %, respectively, were noted in the 1- to 2-year-old age group. However, all the samples were negative for C. pneumoniae by PCR. The study revealed a high seroprevalence of C. pneumoniae in Vietnamese infants and children with HIV/AIDS.

H1N1/09 influenza A virus infection of immortalized first trimester human trophoblast cell lines.

PROBLEM: Although high maternal mortality was reported in the most recent pandemic of swine-origin influenza A H1N1/09 (H1N1/09), its direct effects on the feto-placental unit are unknown. In the present study, we examined the susceptibility of immortalized human trophoblasts to clinical isolates of pandemic 2009 H1N1 influenza A (H1N1/09) virus. METHOD OF STUDY: The H1N1/09 virus was isolated from a patient with influenza, sequenced and identified as the A/Narita/2009 (H1N1) strain. The trophoblast cell lines Swan71 and HTR8 were challenged with the virus and examined for the expression of H1N1/09 viral RNA and proteins. RESULTS: Viral RNA and proteins were observed 24 hr after inoculation. However, viral release was not detected. CONCLUSION: First trimester human trophoblast cell lines were susceptible to the H1N1/09 influenza A virus. However, viral release and cytopathic effects were minimal. Our data suggest that placental damage by the H1N1/09 virus may be limited.

Short communication: Drug resistance mutations in the HIV type 1 protease and reverse transcriptase genes in antiretroviral-naive Vietnamese children.

Anti-HIV drugs have recently become available for the treatment of children infected with HIV in Vietnam; however, the genetic background of HIV-1 drug resistance in antiretroviral-naive children has yet to be studied. Of the 104 HIV-1 CRF01-AE subtype strains that were previously isolated from antiretroviral-naive children from the provinces of southern Vietnam and hospitalized in Children Hospital 1 in Ho Chi Minh City from 2004 to 2005, 79 strains were used for amplification and sequence analyses of the protease and reverse transcriptase (RT) genes. Minor mutations were found in the protease gene, including L10I, I13V, G16E, M36I, D60E, I62V, I64V, L63P, H69K, V82I, and I93L. Of these mutations, M36I and H69K were detected in all of the strains that were studied. However, all of the amino acid changes in the protease gene were considered to be polymorphisms. In the RT gene, three major mutations were detected in six strains: the V75M mutation in one strain, the Y181C mutation in two strains, and the M184I mutation in three strains. The prevalence of primary or transmitted HIV drug resistance to all of the drugs and drug classes that were evaluated in this study was 7.6%. These findings provide a useful background for antiretroviral therapy in Vietnam and contribute reference data for the surveillance of HIV drug resistance around the world. This study suggests that the prevalence of HIVDR in Vietnam may have recently increased. The monitoring of HIV drug resistance in Vietnam is necessary.

The therapeutic potential of the recombinant antigen from Dirofilaria immitis (rDiAg) for immune-mediated pregnancy loss.

The mammalian fetuses are semi-allograft for mothers. Therefore the failure of immunological tolerance often causes pregnancy loss. Recently, the effects of helminthes therapy for immune mediated diseases have been reported. In the present study we employed the murine model to examine the therapeutic potential of the recombinant antigen from a nematoda parasite, Dirofilaria immitis for immune mediated pregnancy loss. Recombinant D. immitis polyproteins (rDiAg) had been cloned and selected by us for the strongest immuno-regulatory activities in parasite antigens. Female CBA/J mice were injected with sterilized rDiAg or PBS solution using micro-osmotic pumps before mating. Pregnant CBA/J mice were sacrificed on day 13.5 for scoring the number of resorbed and viable fetuses for histological and immunological analysis. The serum cytokine concentrations were measured using suspension array system. The resorption rate of mock-treated mice was 42.9% (resorbed fetus 12/total fetus 28). The resorption rate was decreased to 11.1% (resorbed fetus 3/total fetus 27) with rDiAg treatments. The IL-4, IL-23 and TNF-α concentrations in serum were significantly lower in rDiAg-treated mice than mock-treated mice. The serum IL-17 level was also reduced in rDiAg-treated mice but the difference was not significant. The rDiAg treatment reduced the resorption rates of CBA/J×DBA/2J mouse model, which mimic human pregnancy failures with allo-immune backgrounds. Our observations suggest as the first time of therapeutic potentials of the rDiAg for pregnancy loss.

Lipopolysaccharide (LPS)-induced Interferon (IFN)-gamma production by decidual mononuclear cells (DMNC) is interleukin (IL)-2 and IL-12 dependent.

PROBLEM: Th1-shifted immune response is believed to be harmful for successful pregnancy because of activation of maternal cytotoxic T lymphocytes and natural killer cells. However, its effects on Toll-like receptor (TLR)-mediated innate immune response are so far unknown and this study has been undertaken to address the issue. METHOD OF STUDY: decidual tissues were obtained from 16 pregnant women undergoing elective termination during the first trimester pregnancy for socioeconomic reasons. Decidual Mononuclear Cells (DMNC) were stimulated with suboptimal doses of IL-2 and IL-12 with/without LPS, considered to be a TLR4 ligand, for 48 hr. Productions of IFN-γ and tumor necrosis factor (TNF)-α in culture supernatant were measured with ELISA. RESULTS: (i) IFN-γ production was induced with LPS alone which was strongly up-regulated in the presence of IL-2 and IL-12. (ii) TNF-α was also induced by LPS but was not affected by the presence of IL-2 and IL-12. CONCLUSION: IL-2 and IL-12 up-regulated the production of IFN-γ in DMNC through increasing their susceptibility to LPS. TNF-α production is independent of such a mechanism.

H3N2 influenza A virus replicates in immortalized human first trimester trophoblast cell lines and induces their rapid apoptosis.

PROBLEM: Epidemiological data suggested that pandemic influenza increased the risks of spontaneous abortion and premature labor, while seasonal influenza also increased the risk of schizophrenia in adolescence. However, their pathogenesis is so far unknown. METHOD OF STUDY: The first trimester trophoblast cell lines, namely, Swan71 and HTR8 cells were challenged with A/Udorn/72 influenza virus (H3N2). At indicated time points, cells were examined for expression of influenza proteins. Viral replication in culture media, apoptosis and the expression of human leukocyte antigen (HLA)-G were also examined. RESULTS: Intracellular localization of viral proteins was observed. Twenty-four hours after inoculation, virus was detected in culture media while most cells fell into apoptosis. During apoptosis, expression of HLA-G was unchanged. CONCLUSION: We revealed replication of low pathogenic influenza virus in the first trimester trophoblast cell lines. Placental damages are likely to be induced by direct cytopathic effects of influenza virus and subsequent apoptosis rather than down regulation of HLA-G expression and subsequent rejection by maternal immune system.

Postinfection passive transfer of KD-247 protects against simian/human immunodeficiency virus-induced CD4+ T-cell loss in macaque lymphoid tissue.

BACKGROUND: Preadministration of high-affinity humanized anti-HIV-1 mAb KD-247 by passive transfer provides sterile protection of monkeys from heterologous chimeric simian/human immunodeficiency virus infection. METHODS: Beginning 1 h, 1 day, or 1 week after simian/human immunodeficiency virus-C2/1 challenge (20 50% tissue culture infective dose), mature, male cynomolgus monkeys received multiple passive transfers of KD-247 (45 mg/kg) on a weekly basis for approximately 2 months. Concentrations and viral loads were measured in peripheral blood, and CD4 T-cell counts were examined in both peripheral blood and various lymphoid tissues. RESULTS: Pharmacokinetic examination revealed similar plasma maintenance levels ranging from 200 to 500 microg/ml of KD-247 in the three groups. One of the six monkeys given KD-247 could not maintain these concentrations, and elicitation of anti-KD-247 idiotype antibody was suggested. All monkeys given KD-247 exhibited striking postinfection protection against both CD4 T-cell loss in various lymphoid tissues and atrophic changes in organs compared with control group animals treated with normal human immunoglobulin G. The KD-247-treated groups were also partially protected against plasma viral load elevation in peripheral blood samples, although the complete protection previously reported with preadministration of this mAb was not achieved. CONCLUSION: Postinfection passive transfer of humanized mAb KD-247 with strong neutralizing capacity against challenged virus simian/human immunodeficiency virus-C2/1 protected CD4 T cells in lymphoid organs.

Subtyping and env C2/V3 sequence analysis of HIV-1 isolated from HIV-infected children hospitalized in Children Hospital 1, Vietnam during 2004-2005.

A molecular epidemiological study was conducted on 104 HIV-1 strains isolated from HIV-infected children hospitalized in Children Hospital 1 in Ho Chi Minh City, Vietnam during 2004-2005. Genetic subtyping based on env C2/V3 sequences revealed that CRF01-AE was the sole circulating recombinant form found in this study. Sequence analysis of the V3 loop showed that GPGQ tetramer was the most common V3 loop core motif identified in the HIV-1 strains studied (89.5%). The findings raise great concern about HIV-infected children in Vietnam and provide up-to-date molecular epidemiological information of HIV-1 circulating in Vietnam during the study period.

Chimeric adenovirus type 5/35 vector encoding SIV gag and HIV env genes affords protective immunity against the simian/human immunodeficiency virus in monkeys.

Replication-defective adenovirus type 5 (Ad5) vector-based vaccines are widely known to induce strong immunity against immunodeficiency viruses. To exploit this immunogenicity while overcoming the potential problem of preexisting immunity against human adenoviruses type 5, we developed a recombinant chimeric adenovirus type 5 with type 35 fiber vector (rAd5/35). We initially produced a simian immunodeficiency virus (SIV) gag DNA plasmid (rDNA-Gag), a human immunodeficiency virus type 1 (HIV-1) 89.6 env DNA plasmid (rDNA-Env) and a recombinant Ad5/35 vector encoding the SIV gag and HIV env gene (rAd5/35-Gag and rAd5/35-Env). Prime-boost vaccination with rDNA-Gag and -Env followed by high doses of rAd5/35-Gag and -Env elicited higher levels of cellular immune responses than did rDNAs or rAd5/35s alone. When challenged with a pathogenic simian human immunodeficiency virus (SHIV), animals receiving a prime-boost regimen or rAd5/35s alone maintained a higher number of CD4(+) T cells and remarkably suppressed plasma viral RNA loads. These findings suggest the clinical promise of an rAd5/35 vector-based vaccine.

Anti-V3 humanized antibody KD-247 effectively suppresses ex vivo generation of human immunodeficiency virus type 1 and affords sterile protection of monkeys against a heterologous simian/human immunodeficiency virus infection.

In an accompanying report (Y. Eda, M. Takizawa, T. Murakami, H. Maeda, K. Kimachi, H. Yonemura, S. Koyanagi, K. Shiosaki, H. Higuchi, K. Makizumi, T. Nakashima, K. Osatomi, S. Tokiyoshi, S. Matsushita, N. Yamamoto, and M. Honda, J. Virol. 80:5552-5562, 2006), we discuss our production of a high-affinity humanized monoclonal antibody, KD-247, by sequential immunization with V3 peptides derived from human immunodeficiency virus type 1 (HIV-1) clade B primary isolates. Epitope mapping revealed that KD-247 recognized the Pro-Gly-Arg V3 tip sequence conserved in HIV-1 clade B isolates. In this study, we further demonstrate that in vitro, KD-247 efficiently neutralizes CXCR4- and CCR5-tropic primary HIV-1 clade B and clade B' with matching neutralization sequence motifs but does not neutralize sequence-mismatched clade B and clade E isolates. Monkeys were provided sterile protection against heterologous simian/human immunodeficiency virus challenge by the passive transfer of a single high dose (45 mg per kg of body weight) of KD-247 and afforded partial protection by lower antibody doses (30 and 15 mg per kg). Protective neutralization endpoint titers in plasma at the time of virus challenge were 1:160 in animals passively transferred with a high dose of the antibody. The antiviral efficacy of the antibody was further confirmed by its suppression of the ex vivo generation of primary HIV-1 quasispecies in peripheral blood mononuclear cell cultures from HIV-infected individuals. Therefore, KD-247 promises to be a valuable tool not only as a passive immunization antibody for the prevention of HIV infection but also as an immunotherapy for the suppression of HIV in phenotype-matched HIV-infected individuals.

Induction of positive cellular and humoral immune responses by a prime-boost vaccine encoded with simian immunodeficiency virus gag/pol.

It is believed likely that immune responses are responsible for controlling viral load and infection. In this study, when macaques were primed with plasmid DNA encoding SIV gag and pol genes (SIVgag/pol DNA) and then boosted with replication-deficient vaccinia virus DIs recombinant expressing the same genes (rDIsSIVgag/pol), this prime-boost regimen generated higher levels of Gag-specific CD4+ and CD8+ T cell responses than did either SIVgag/pol DNA or rDIsSIVgag/pol alone. When the macaques were i.v. challenged with pathogenic simian/HIV, the prime-boost group maintained high CD4+ T cell counts and reduced plasma viral loads up to 30 wk after viral challenge, whereas the rDIsSIVgag/pol group showed only a partial attenuation of the viral infection, and the group immunized with SIVgag/pol DNA alone showed none at all. The protection levels were better correlated with the levels of virus-specific T cell responses than the levels of neutralization Ab responses. These results demonstrate that a vaccine regimen that primes with DNA and then boosts with a replication-defective vaccinia virus DIs generates anti-SIV immunity, suggesting that it will be a promising vaccine regimen for HIV-1 vaccine development.

Priming-boosting vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin and a nonreplicating vaccinia virus recombinant leads to long-lasting and effective immunity.

Virus-specific T-cell responses can limit immunodeficiency virus type 1 (HIV-1) transmission and prevent disease progression and so could serve as the basis for an affordable, safe, and effective vaccine in humans. To assess their potential for a vaccine, we used Mycobacterium bovis bacillus Calmette-Guérin (BCG)-Tokyo and a replication-deficient vaccinia virus strain (DIs) as vectors to express full-length gag from simian immunodeficiency viruses (SIVs) (rBCG-SIVgag and rDIsSIVgag). Cynomolgus macaques were vaccinated with either rBCG-SIVgag dermally as a single modality or in combination with rDIsSIVgag intravenously. When cynomologus macaques were primed with rBCG-SIVgag and then boosted with rDIsSIVgag, high levels of gamma interferon (IFN-gamma) spot-forming cells specific for SIV Gag were induced. This combination regimen elicited effective protective immunity against mucosal challenge with pathogenic simian-human immunodeficiency virus for the 1 year the macaques were under observation. Antigen-specific intracellular IFN-gamma activity was similarly induced in each of the macaques with the priming-boosting regimen. Other groups receiving the opposite combination or the single-modality vaccines were not effectively protected. These results suggest that a recombinant M. bovis BCG-based vector may have potential as an HIV/AIDS vaccine when administered in combination with a replication-deficient vaccinia virus DIs vector in a priming-boosting strategy.

Intravenous inoculation of replication-deficient recombinant vaccinia virus DIs expressing simian immunodeficiency virus gag controls highly pathogenic simian-human immunodeficiency virus in monkeys.

To be effective, a vaccine against human immunodeficiency virus type 1 (HIV-1) must induce virus-specific T-cell responses and it must be safe for use in humans. To address these issues, we developed a recombinant vaccinia virus DIs vaccine (rDIsSIVGag), which is nonreplicative in mammalian cells and expresses the full-length gag gene of simian immunodeficiency virus (SIV). Intravenous inoculation of 10(6) PFU of rDIsSIVGag in cynomologus macaques induced significant levels of gamma interferon (IFN-gamma) spot-forming cells (SFC) specific for SIV Gag. Antigen-specific lymphocyte proliferative responses were also induced and were temporally associated with the peak of IFN-gamma SFC activity in each macaque. In contrast, macaques immunized with a vector control (rDIsLacZ) showed no significant induction of antigen-specific immune responses. After challenge with a highly pathogenic simian-human immunodeficiency virus (SHIV), CD4(+) T lymphocytes were maintained in the peripheral blood and lymphoid tissues of the immunized macaques. The viral set point in plasma was also reduced in these animals, which may be related to the enhancement of virus-specific intracellular IFN-gamma(+) CD8(+) cell numbers and increased antibody titers after SHIV challenge. These results demonstrate that recombinant DIs has potential for use as an HIV/AIDS vaccine.

Higher levels of IL-18 circulate during primary infection of monkeys with a pathogenic SHIV than with a nonpathogenic SHIV.

We have monitored kinetics of peripheral blood Interleukin (IL)-18 level, viral RNA load, and CD4(+) T cell counts in cynomolgus and rhesus macaques following infections of various simian/human immunodeficiency viruses (SHIVs) causing differential pathogenicity. Infections of cynomolgus and rhesus macaques with pathogenic SHIVs-C2/1 and -89.6PD, respectively, induced high levels of plasma IL-18 (0.1-1 ng/ml) and enhanced apoptosis of peripheral blood T cells during primary viremia, along with a rapid decline of CD4(+) T cells and a high level of set point viral load after primary viremia (six of six cases). In contrast, infections of cynomolgus macaques with nonpathogenic SHIVs-TH09V3 and -MD14 did not cause such IL-18 elevation, showing no decline of CD4(+) T cells and no or low viral set point level following primary viremia (three of three cases). Thus, the elevation of circulating IL-18 level during primary viral infection can be a good indicator of an active pathogenic viral infection. However, the role of increased IL-18 remains to be elucidated and needs further investigation.

Structural analysis of vaccinia virus DIs strain: application as a new replication-deficient viral vector.

DIs is a restrictive host range mutant of vaccinia virus strain DIE that grows well only in chick embryo fibroblast cells but is unable to grow in most mammalian cells. In this study, we identified one major deletion (15.4 kbp) which results in the loss of 19 putative open reading frames in the left end of the genome. We then established a system to express foreign genes by inserting them into the deleted region of DIs. We constructed rDIs to express the bacteriophage T7 polymerase (T7pol) gene and showed the expression in various mammalian cell lines by reporter luciferase gene expression under the T7 promoter. We also expressed the full-length human immunodeficiency virus (HIV)-1 NL432 gag gene. The expressed gag gene product induced high levels of cytotoxic T lymphocytes in immunized mice. These data suggest that DIs is useful as an efficient, transient replication-deficient viral vector.