[Show abstract][Hide abstract] ABSTRACT: We perform whole transcriptome analysis of distinct CD8+ T-cell memory populations arising endogenously following immunization with clinical and pre-clinical vaccines regimens.•We find that transcriptional proximity relationships between endogenous TN, TCM, TEM and TEFF place memory cells at an intermediate state of differentiation between TN and TEFF.•We compare transcriptional differences between TN, TCM, TEM and TEFF cells with known changes when CD8+ T cells undergo repeated antigen stimulation.•We find that at a single time-point following vaccination, cells can be found with the transcriptional imprint of distinct exposures to antigen.•Our analysis favors the progressive differentiation model whereby cumulative antigen stimulation drives differentiation specifically from TN > TCM > TEM > TEFF.
[Show abstract][Hide abstract] ABSTRACT: A humanized neutralizing antibody, KD-247, targets the V3 loop of HIV-1 Env. HIV-1 bearing the GPGR sequence at the V3 loop is potentially susceptible to KD-247. However, not all GPGR-positive HIV-1 isolates are neutralized by KD-247. We examined the potential mechanism by which the susceptibility of HIV-1 to KD-247-mediated neutralization is regulated.
We searched for nonepitope neutralization regulatory (NNR) mutations that sensitize GPGR-bearing HIV-1AD8 to KD-247 and mapped the locations of such mutations relative to the V3 loop.
: We generated a functional HIV-1AD8 Env library, and evaluated the viral susceptibility to KD-247 by measuring the half-inhibitory concentration (IC50) to KD-247 on TZM-bl cell assay.
We identified nine KD-247-sensitizing NNR mutations from 30 mutations in various regions of gp120, including the V1/V2 loop, C2, V3 loop, C4, and C5. They specifically affected KD-247-mediated neutralization, as they did not affect the b12-mediated neutralization. When combined, the KD-247-sensitizing NNR mutations additively sensitized the virus to KD-247 by up to 10 000 folds. The KD-247-sensitizing NNR mutations increased KD-247 binding to the virion. Notably, the NNR mutation in C4 coincides with the CD4-binding site of gp120.
Given that most of the KD-247-sensitizing NNR mutations are remote from V3 loop, it is reasonable to hypothesize that the steady-state, local conformation of the V3 loop is regulated by the interdomain contact of gp120. Our mutational analysis complements crystallographic studies by helping provide a better understanding of the steady-state conformation and the functional geometry of Env.
AIDS (London, England) 08/2011; 25(18):2209-16. · 6.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.
Proceedings of the National Academy of Sciences 03/2011; 108(14):5724-9. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only tuberculosis (TB) vaccine currently available, but its efficacy against adult pulmonary TB remains controversial. BCG induces specific immune responses to mycobacterial antigens and may elicit protective immunity against TB. TB remains a major public health problem, especially among the elderly, yet the efficacy of BCG in the elderly is unknown. We investigated the ability of BCG vaccination to prevent TB in young (6-week-old), middle-aged (18-month-old), and old (60-month-old) guinea pigs. BCG-Tokyo vaccination reduced the growth of Mycobacterium tuberculosis H37Rv in all three groups. By use of an enzyme-linked immunospot (ELISPOT) assay, antigen-specific gamma interferon (IFN-γ)-producing cells were detected in the 60-month-old guinea pigs after a booster vaccination with BCG-Tokyo. Our findings suggest that BCG-Tokyo has a protective effect against tuberculosis infection regardless of age.
[Show abstract][Hide abstract] ABSTRACT: Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8(+) cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8(+) T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D(d) better than the native sequences, and clones with distinct specificities were elicited by alternative vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8(+) T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.
The Journal of Immunology 09/2009; 183(4):2425-34. · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
AIDS (London, England) 07/2009; 23(12):1485-94. · 6.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, we found that the electric potential derived from the redox reaction of ultraviolet (UV)-illuminated CD4-conjugated titanium dioxide (TiO2) inactivated a wide range of high-titered primary HIV-1 isolates, regardless of virus co-receptor usage or genetic clade. In vitro incubation of HIV-1 isolates with CD4-conjugated TiO2 (CD4-TiO2) followed by UV illumination led to inhibition of viral infectivity in both H9 cells and peripheral blood mononuclear cells as well as to the complete inactivation of plasma virions from HIV-1-infected individuals. Treatment with a newly established extra-corporeal circulation system with the photocatalyst in rhesus macaques completely inactivated plasma virus in the system and effectively reduced the infectious plasma viral load. Furthermore, plasma viremia and infectious viral loads were controlled following a second therapeutic photocatalyst treatment during primary SIV(mac239) infection of macaques. Our findings suggest that this therapeutic immunophysical strategy may help control human immunodeficiency viral infection in vivo.
Journal of Medical Virology 09/2008; 80(8):1322-31. · 2.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The functional human immune system, including T, B, and natural killer lymphocytes, is reconstituted in NOD/Shi-scid/IL-2Rgamma(null) (NOG) mice that receive hematopoietic stem cell transplants. Here, we show that these humanized mice can recapitulate key aspects of Epstein-Barr virus (EBV) infection in humans. Inoculation with approximately 1 x 10(3) TD(50) (50% transforming dose) of EBV caused B cell lymphoproliferative disorder, with histopathological findings and latent EBV gene expression remarkably similar to that in immunocompromised patients. Inoculation with a low dose of virus (<or=1 x 10(1) TD(50)), in contrast, resulted in apparently asymptomatic persistent infection. Levels of activated CD8(+) T cells increased dramatically in the peripheral blood of infected mice, and enzyme-linked immunospot assay and flow cytometry demonstrated an EBV-specific T cell response. Immunoglobulin M antibody specific to the EBV-encoded protein BFRF3 was detected in serum from infected mice. The NOG mouse is the most comprehensive small-animal model of EBV infection described to date and should facilitate studies of the pathogenesis, prevention, and treatment of EBV infection.
The Journal of Infectious Diseases 09/2008; 198(5):673-82. · 5.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epstein-Barr virus (EBV) causes EBV-associated lymphoproliferative diseases in patients with profound immune suppression. Most of these diseases are life-threatening and the prognosis of AIDS-associated lymphomas is extremely unfavorable. Polyclonal expansion of virus infected B-cell predisposes them to transformation. We investigated the possibility of nuclear factor kappa B (NF-kappaB) inhibition by dehydroxymethylepoxyquinomicin (DHMEQ) for the treatment and prevention of EBV-associated lymphoproliferative diseases. We examined the effect of DHMEQ on apoptosis induction in four EBV-transformed lymphoblastoid cell lines as well as peripheral blood mononuclear cells infected with EBV under immunosuppressed condition. DHMEQ inhibits NF-kappaB activation in EBV-transformed lymphoblastoid cell lines and induces apoptosis by activation of mitochondrial and membranous pathways. Using an in vivo NOD/SCIDgammac mouse model, we showed that DHMEQ has a potent inhibitory effect on the growth of lymphoblastoid cells. In addition, DHMEQ selectively purges EBV-infected cells expressing latent membrane protein (LMP) 1 from peripheral blood mononuclear cells and inhibits the outgrowth of lymphoblastoid cells. These results suggest that NF-kappaB is a molecular target for the treatment and prevention of EBV-associated lymphoproliferative diseases. As a potent NF-kappaB inhibitor, DHMEQ is a potential compound for applying this strategy in clinical medicine.
Microbes and Infection 07/2008; 10(7):748-56. · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) utilizes the macromolecular machinery of the infected host cell to produce progeny virus. The discovery of cellular factors that participate in HIV-1 replication pathways has provided further insight into the molecular basis of virus-host cell interactions. Here, we report that the suppressor of cytokine signaling 1 (SOCS1) is an inducible host factor during HIV-1 infection and regulates the late stages of the HIV-1 replication pathway. SOCS1 can directly bind to the matrix and nucleocapsid regions of the HIV-1 p55 Gag polyprotein and enhance its stability and trafficking, resulting in the efficient production of HIV-1 particles via an IFN signaling-independent mechanism. The depletion of SOCS1 by siRNA reduces both the targeted trafficking and assembly of HIV-1 Gag, resulting in its accumulation as perinuclear solid aggregates that are eventually subjected to lysosomal degradation. These results together indicate that SOCS1 is a crucial host factor that regulates the intracellular dynamism of HIV-1 Gag and could therefore be a potential new therapeutic target for AIDS and its related disorders.
Proceedings of the National Academy of Sciences 02/2008; 105(1):294-9. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In a previous study, we demonstrated that humanized NOD/SCID/IL2Rgamma(null) (hNOG) mice constructed with human hematopoietic stem cells (HSCs) allow efficient human immunodeficiency virus type 1 (HIV-1) infection. However, HIV-1 infection could be monitored for only 43 days in the animals due to their short life spans. By transplanting HSCs without any myeloablation methods, the mice successfully survived longer than 300 days with stable engraftment of human cells. The mice showed high viremia state for more than the 3 months examined, with systemic HIV-1 infection and gradual decrease of CD4+ T cells analogous to that in humans. These capacities of the hNOG mice are very attractive for modeling mechanisms of AIDS progression and therapeutic strategy.
Journal of Virology 01/2008; 81(23):13259-64. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Abstract A collaborative group for studying vertical transmission of human immunodeficiency virus (HIV)-1 in pregnant women and their babies was established in Japan in 1989. Forty-two infants, including 13 HIV-1-infected, 25 uninfected and four of undetermined status and 15 control children born to HIV-1 negative mothers were diagnosed and followed from birth to 1.5 years. All strains from HIV-positive infants were either clade E (eight infants, 61.5%) or B (five infants, 38.5%) according to DNA sequencing specific for the HIV-1 C2-V3 region. The 42 mothers with HIV-1 were women with sexual-risk behavior from all regions, but were concentrated in the Kanto District. In this group of HIV-infected children, there was no significant difference between the transmissibility of their mother's clade E and B viruses. Eight (61.5%) of the 13 virus-infected babies were Japanese and five (62.5%) of the eight were positive for HIV-1 clade E. The V3 loop region of the clade E virus of the babies was conserved but approximately 60% of the sequences which showed a substitution of aspartic acid by asparagine at position 29. The results suggest that HIV-1 clade E may be predominant in vertical transmissions and are phenotypically different from HIV-1 in persons with various other risk behaviors in Japan.
Pediatrics International 10/2007; 40(5):503 - 509. · 0.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Infectious factors in breast milk such as viral particles and living infected cells are of prime importance in the transmission of HIV by breastfeeding.
To perform effective approaches for reducing HIV transmission via breastfeeding, we investigated the biological importance of infectious viral particles and infected BMCs in breast milk.
Alteration of viral infectivity was monitored using a modified experimental infection assay that exploited the cytotoxicity of breast milk, and BMC viability was evaluated by flow-cytometric analysis.
Infectious viral particles were found to decrease time-dependently after contact with breast milk, whereas BMCs showed prolonged survival in breast milk.
The biological importance of infected BMCs in breast milk for the transmission of HIV via breastfeeding was considered.
Journal of Clinical Virology 08/2007; 39(3):222-5. · 3.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Constitutive nuclear factor kappaB (NF-kappaB) activation characterizes Hodgkin/Reed-Sternberg (H-RS) cells. Blocking constitutive NF-kappaB has been shown to be a potential strategy to treat Hodgkin lymphoma (HL). Here, for the first time we show that although constitutive NF-kappaB level of H-RS cell lines is very high, topoisomerase inhibitors further enhance NF-kappaB activation through IkappaB kinase activation in not only H-RS cell lines with wild-type IkappaBalpha, but also in those with IkappaBalpha mutations and lacking wild-type IkappaBalpha. Thus, both constitutive and inducible NF-kappaB are potential targets to treat HL. We also present the data that indicate the involvement of IkappaBbeta in NF-kappaB induction by topoisomerase inhibitors. A new NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) inhibited constitutive NF-kappaB activity and induced apoptosis of H-RS cell lines. DHMEQ also inhibited the growth of H-RS cells without significant systemic toxicity in a NOD/SCID/gammac(null) (NOG) mice model. DHMEQ and topoisomerase inhibitors revealed enhancement of apoptosis of H-RS cells by blocking inducible NF-kappaB. Results of this study suggest that both constitutive and inducible NF-kappaB are molecular targets of DHMEQ in the treatment of HL. The results also indicate that IkappaBbeta is involved in NF-kappaB activation in H-RS cells and IkappaBbeta substitutes for IkappaBalpha in H-RS cells lacking wild-type IkappaBalpha.
[Show abstract][Hide abstract] ABSTRACT: Critical to the development of an effective HIV/AIDS model is the production of an animal model that reproduces long-lasting active replication of HIV-1 followed by elicitation of virus-specific immune responses. In this study, we constructed humanized nonobese diabetic/severe combined immunodeficiency (NOD/SCID)/interleukin-2 receptor gamma-chain knockout (IL2Rgamma(null)) (hNOG) mice by transplanting human cord blood-derived hematopoietic stem cells that eventually developed into human B cells, T cells, and other monocytes/macrophages and 4 dendritic cells associated with the generation of lymphoid follicle-like structures in lymphoid tissues. Expressions of CXCR4 and CCR5 antigens were recognized on CD4+ cells in peripheral blood, the spleen, and bone marrow, while CCR5 was not detected on thymic CD4+ T cells. The hNOG mice showed marked, long-lasting viremia after infection with both CCR5- and CXCR4-tropic HIV-1 isolates for more than the 40 days examined, with R5 virus-infected animals showing high levels of HIV-DNA copies in the spleen and bone marrow, and X4 virus-infected animals showing high levels of HIV-DNA copies in the thymus and spleen. Furthermore, we detected both anti-HIV-1 Env gp120- and Gag p24-specific antibodies in animals showing a high rate of viral infection. Thus, the hNOG mice mirror human systemic HIV infection by developing specific antibodies, suggesting that they may have potential as an HIV/AIDS animal model for the study of HIV pathogenesis and immune responses.
[Show abstract][Hide abstract] ABSTRACT: The prevalence of adult HIV/AIDS in Thailand is declining due to intense prevention strategies, but it still continues to be a critical health problem with a prevalence of 1.5%. Several HIV vaccine candidates for the prevention of HIV infection or progress to AIDS were examined in clinical trials. We evaluated the cost-effectiveness of a vaccination regimen (rBCG prime-rDIs boost) currently in its pre-clinical phase. The cost-effectiveness of three interventions (vaccination, highly active antiretroviral treatment [HAART], and the combination of the two) through an existing vaccination program was assessed in a Markov model. The disability-adjusted life year (DALY) was the main effectiveness measure. In the base case the efficacy of the vaccine for preventing HIV infection was assumed to be 30%. The cost of the vaccine was estimated on the basis of its predicted production capacities in Thailand. The incremental cost-effectiveness ratios of vaccination, HAART, and the combination were about dollar US 75, dollar US 610, and dollar US 267 per DALY averted compared with the do-nothing strategy in the base case. The HAART-only strategy seemed to be less cost-effective than the other options under the current assumptions. Sensitivity analyses indicated that the new HIV infection rate and the vaccine efficacy could affect the results.
Japanese journal of infectious diseases 07/2006; 59(3):168-73. · 1.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Virology 07/2006; 80(11):5563-70. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An antibody response capable of neutralizing not only homologous but also heterologous forms of the CXCR4-tropic human immunodeficiency virus type 1 (HIV-1) MNp and CCR5-tropic primary isolate HIV-1 JR-CSF was achieved through sequential immunization with a combination of synthetic peptides representing HIV-1 Env V3 sequences from field and laboratory HIV-1 clade B isolates. In contrast, repeated immunization with a single V3 peptide generated antibodies that neutralized only type-specific laboratory-adapted homologous viruses. To determine whether the cross-neutralization response could be attributed to a cross-reactive antibody in the immunized animals, we isolated a monoclonal antibody, C25, which neutralized the heterologous primary viruses of HIV-1 clade B. Furthermore, we generated a humanized monoclonal antibody, KD-247, by transferring the genes of the complementary determining region of C25 into genes of the human V region of the antibody. KD-247 bound with high affinity to the "PGR" motif within the HIV-1 Env V3 tip region, and, among the established reference antibodies, it most effectively neutralized primary HIV-1 field isolates possessing the matching neutralization sequence motif, suggesting its promise for clinical applications involving passive immunizations. These results demonstrate that sequential immunization with B-cell epitope peptides may contribute to a humoral immune-based HIV vaccine strategy. Indeed, they help lay the groundwork for the development of HIV-1 vaccine strategies that use sequential immunization with biologically relevant peptides to overcome difficulties associated with otherwise poorly immunogenic epitopes.
Journal of Virology 07/2006; 80(11):5552-62. · 4.65 Impact Factor