Diminished matrix metalloproteinase 9 secretion in human immunodeficiency virus-infected mononuclear phagocytes: modulation of innate immunity and implications for neurological disease.

Neurotoxic secretory products from virus-infected mononuclear phagocytes (MP; perivascular macrophages and microglia) orchestrate the neuropathogenesis of human immunodeficiency virus type one (HIV-1) infection. To uncover such MP products and their relationship to disease, we used a proteomics platform consisting of one dimensional polyacrylamide gel electrophoresis (1-DE), mass spectrometry peptide sequencing, and bioinformatics in order to identify from HIV-1-infected monocyte-derived macrophages (MDM) secretions. Matrix metalloproteinase 9 (MMP 9) secreted in abundance in MDM was markedly down-regulated following viral infection. A negative correlation between MMP 9 and HIV-1 reverse transcriptase activity was shown by quantitative Western blot assays. These data further demonstrate immunoregulatory activities of HIV-1-infected MDM providing unique insights into cellular function in disease.

Infection of a chimeric simian and human immunodeficiency virus with CCR5-specific HIV-1 envelope to Rhesus macaques.

Human immunodeficiency virus (HIV) infects lymphocytes and macrophages via CD4 and chemokine receptors. In this study, the infectivity of a chimeric simian and human immunodeficiency virus (SHIV) having a CCR5-specific HIV-1 envelope gene was examined. A SHIV strain termed SHIV-JRFL could enter cells via CD4 with a chemokine receptor CCR5, not CXCR4, and the viral replication was suppressed by recombinant human RANTES, one of beta-chemokines. The intravenous inoculation of SHIV-JRFL into two rhesus macaques resulted in a systemic infection, though it was rather weak. During the early infection, the production of RANTES from Con A-stimulated PBMCs of the infected monkeys increased. These results suggested that beta-chemokine has the potential to limit the infectivity of an R5-type SHIV.

Genomic and proteomic microglial profiling: pathways for neuroprotective inflammatory responses following nerve fragment clearance and activation.

Microglia, a primary immune effector cell of the central nervous system (CNS) affects homeostatic, neuroprotective, regenerative and degenerative outcomes in health and disease. Despite these broad neuroimmune activities linked to specific environmental cues, a precise cellular genetic profile for microglia in the context of disease and repair has not been elucidated. To this end we used nucleic acid microarrays, proteomics, immunochemical and histochemical tests to profile microglia in neuroprotective immune responses. Optic and sciatic nerve (ON and SN) fragments were used to stimulate microglia in order to reflect immune consequences of nervous system injury. Lipopolysaccharide and latex beads-induced microglial activation served as positive controls. Cytosolic and secreted proteins were profiled by surface enhanced laser desorption ionization-time of flight (SELDI-TOF) ProteinChip, 1D and 2D difference gel electrophoresis. Proteins were identified by peptide sequencing with tandem mass spectrometry, ELISA and western blot tests. Temporal expression of pro-inflammatory cytokines, antioxidants, neurotrophins, and lysosomal enzyme expression provided, for the first time, a unique profile of secreted microglia proteins with neuroregulatory functions. Most importantly, this molecular and biochemical signature supports a broad range of microglial functions for debris clearance and promotion of neural repair after injury.

DNA vaccination of macaques by a full-genome SHIV plasmid that has an IL-2 gene and produces non-infectious virus particles.

We previously reported that a mutant full-sized plasmid DNA vaccine regime in macaques was effective against a homologous challenge [Akahata W, Ido E, Shimada T, Katsuyama K, Yamamoto H, Uesaka H, et al. DNA vaccination of macaques by a full genome HIV-1 plasmid which produces non-infectious virus particles. Virology 2000;275:116-24; Akahata W, Ido E, Akiyama H, Uesaka H, Enose Y, Horiuchi R, et al. DNA vaccination of macaques by a full genome SHIV-1 plasmid that produces non-infectious virus particles. J Gen Virol 2003;84:2237-44]. In this study, to evaluate the DNA vaccination regime against a heterologous challenge, a novel plasmid named pSHIV-ZF1*IL-2 was constructed. Four monkeys were intramuscularly and intradermally injected four times with the pSHIV-ZF1*IL-2. Vaccinated monkeys were intravenously challenged with a highly pathogenic, heterologous SHIV at 11 weeks post vaccination. All the vaccinated monkeys suppressed the challenge virus rapidly under the detectable level by 16 weeks post challenge. One vaccinated monkey was protected from a loss of CD4+ T cells. These results suggest pSHIV-ZF1*IL-2 alone seems partially effective even against a challenge with a heterologous, pathogenic virus.

Rapid dissemination of a pathogenic simian/human immunodeficiency virus to systemic organs and active replication in lymphoid tissues following intrarectal infection.

A better understanding of virological events during the early phase of human immunodeficiency virus 1 (HIV-1) infection is important for development of effective antiviral vaccines. In this study, by using quantitative PCR and an infectious plaque assay, virus distribution and replication were examined in various internal organs of rhesus macaques for almost 1 month after intrarectal inoculation of a pathogenic simian immunodeficiency virus/HIV chimeric virus (SHIV-C2/1-KS661c). At 3 days post-inoculation (p.i.), proviral DNA was detected in the rectum, thymus and axillary lymph node. In lymphoid tissues, infectious virus was first detected at 6 days p.i. and a high level of proviral DNA and infectious virus were both detected at 13 days p.i. By 27 days p.i., levels of infectious virus decreased dramatically, although proviral DNA load remained unaltered. In the intestinal tract, levels of infectious virus detected were much lower than in lymphoid tissues, whereas proviral DNA was detected at the same level as in lymphoid tissues throughout the infection. In the thymus and jejunum, CD4CD8 double-positive T cells were depleted earlier than CD4 single-positive cells. These results show that the virus spread quickly to systemic tissues after mucosal transmission. Thereafter, infectious virus was actively produced in the lymphoid tissues, but levels decreased significantly after the peak of viraemia. In contrast, in the intestinal tract, infectious virus was produced at low levels from the beginning of infection. Moreover, virus pathogenesis differed in CD4 single-positive and CD4CD8 double-positive T cells.

Proteomic fingerprints distinguish microglia, bone marrow, and spleen macrophage populations.

Mononuclear phagocytes (MP; dendritic cells, monocytes, tissue macrophages, and microglia) maintain tissue homeostasis and provide a first line of defense against invading pathogens. In specific circumstances, MPs also induce inflammatory responses and as such affect disease onset and progression. Despite intensive research into MP biology, little is known of the functional and molecular properties of individual MP subtypes. Using a novel proteomics platform, unique protein patterns and protein identities were observed among populations of spleen and bone marrow macrophages and microglia. Cells were obtained from C57BL/6 mice and were cultivated in macrophage colony-stimulating factor. MP subtypes were indistinguishable by morphological or antigenic criteria. Protein profiling by Surface Enhanced Laser Desorption Ionization-Time of Flight (SELDI-TOF) ProteinChip assays with weak cationic exchange chips showed unique MP spectral profiles. Corresponding protein fractions were recovered by high performance liquid chromatography and identified by liquid chromatography tandem mass spectrometry. The results provide a unique means to distinguish microglia from other MP subtypes.

Protective efficacy of nonpathogenic nef-deleted SHIV vaccination combined with recombinant IFN-gamma administration against a pathogenic SHIV challenge in rhesus monkeys.

We previously reported that a nef-deleted SHIV (SHIV-NI) is nonpathogenic and gave macaques protection from challenge infection with pathogenic SHIV-C2/1. To investigate whether IFN-gamma augments the immune response induced by this vaccination, we examined the antiviral and adjuvant effect of recombinant human IFN-gamma (rIFN-gamma) in vaccinated and unvaccinated monkeys. Nine monkeys were vaccinated with nef-deleted nonpathogenic SHIV-NI. Four of them were administered with rIFN-gamma and the other five monkeys were administered with placebo. After the challenge with pathogenic SHIV-C2/1, CD4(+) T-cell counts were maintained similarly in monkeys of both groups, while those of the unvaccinated monkeys decreased dramatically at 2 weeks after challenge. However, the peaks of plasma viral load were reduced to 100-fold in SHIV-NI vaccinated monkeys combined with rIFN-gamma compared with those in SHIV-NI vaccinated monkeys without rIFN-gamma. The peaks of plasma viral load were inversely correlated with the number of SIV Gag-specific IFN-gamma-producing cells. In SHIV-NI-vaccinated monkeys with rIFN-gamma, the number of SIV Gag-specific IFN-gamma-producing cells of PBMCs increased 2-fold compared with those in SHIV-NI-vaccinated monkeys without rIFN-gamma, and the NK activity and MIP-1alpha production of PBMCs were also enhanced. Thus, vaccination of SHIV-NI in combination with rIFN-gamma was more effective in modulating the antiviral immune system into a Th1 type response than SHIV-NI vaccination alone. These results suggest that IFN-gamma augmented the anti-viral effect by enhancing innate immunity and shifting the immune response to Th1.

DNA vaccination of macaques by a full-genome simian/human immunodeficiency virus type 1 plasmid chimera that produces non-infectious virus particles.

A DNA vaccination regime was investigated previously in rhesus macaques using a full-genome human immunodeficiency virus type 1 (HIV-1) plasmid, which, due to mutations in the nucleocapsid (NC) proteins, produced only non-infectious HIV-1 particles (Akahata et al., Virology 275, 116-124, 2000). In that study, four monkeys were injected intramuscularly 14 times with the plasmid. All of them showed immunological responses against HIV-1 and partial protection from challenge with a simian immunodeficiency virus/HIV (SHIV) chimeric virus. To improve this DNA vaccination regime, the plasmid used for vaccination was changed. In the present study, four macaques were injected intramuscularly eight times with a full-genome SHIV plasmid that produces non-infectious SHIV particles. CTL activities were higher than those observed in monkeys vaccinated previously with the HIV-1 plasmid. In all macaques vaccinated, peak plasma virus loads after homologous challenge with SHIV were two to three orders of magnitude lower than those of the naive controls, and virus loads fell below the level of detection at 6 weeks post-challenge. This suggested that the vaccination regime in this study was partially effective and better than the previous regime.

Protection by intranasal immunization of a nef-deleted, nonpathogenic SHIV against intravaginal challenge with a heterologous pathogenic SHIV.

An effective vaccine against sexual transmission of human immunodeficiency virus (HIV) should elicit both systemic and mucosal immune responses. In this study, to examine the possibility of using an attenuated virus for mucosal immunization, four female macaques were intranasally or intravenously administered with a chimeric simian-human immunodeficiency virus with a deleted nef gene (SHIV-dn). Although all the monkeys had anti-HIV-1 antibodies with neutralizing activity in the plasma, the intranasally immunized monkeys had much higher levels of HIV-1 Env-specific IgG and IgA antibodies in mucosal secretions compared with the intravenously immunized monkeys. Moreover, three of four intranasally immunized monkeys were completely protected from intravaginal challenge with a pathogenic virus, SHIV-89.6P, whereas only one intravenously immunized monkey was protected. Thus, intranasal immunization of an attenuated virus can induce the protective efficacy against intravaginal infection.

Induction of HIV-specific antibody response and protection against vaginal SHIV transmission by intranasal immunization with inactivated SHIV-capturing nanospheres in macaques.

We have previously reported that concanavalin A-immobilized polystyrene nanospheres (Con A-NS) could efficiently capture HIV-1 particles and that intranasal immunization with inactivated HIV-1-capturing nanospheres (HIV-NS) induced vaginal anti-HIV-1 IgA antibody response in mice. In this study, to evaluate the protective effect of immunization, each three macaques was intranasally immunized with Con A-NS or inactivated simian/human immunodeficiency virus KU-2-capturing nanospheres (SHIV-NS) and then intravaginally challenged with a pathogenic virus, SHIV KU-2. After a series of six immunizations, vaginal anti-HIV-1 gp120 IgA and IgG antibodies were detected in all SHIV-NS-immunized macaques. After intravaginal challenge, one of the three macaques in each of the Con A-NS- and SHIV-NS-immunized groups was infected. Plasma viral RNA load of infected macaque in SHIV-NS-immunized macaques was substantially less than that in unimmunized control macaque and reached below the detectable level. However, it could not be determined whether intranasal immunization with SHIV-NS is effective in giving complete protection against intravaginal challenge. To explore the effect of the SHIV-NS vaccine, the remaining non-infected macaques were rechallenged intravenously with SHIV KU-2. After intravenous challenge, all macaques became infected. However, SHIV-NS-immunized macaques had lower viral RNA loads and higher CD4(+) T cell counts than unimmunized control macaques. Plasma anti-HIV-1 gp120 IgA and IgG antibodies were induced more rapidly in the SHIV-NS-immunized macaques than in the controls. The rapid antibody responses having neutralizing activity might contribute to the clearance of the challenge virus. Thus, SHIV-NS-immunized macaques exhibited partial protection to vaginal and systemic challenges with SHIV KU-2.