Selective induction of cell-mediated immunity and protection of rhesus macaques from chronic SHIV(KU2) infection by prophylactic vaccination with a conserved HIV-1 envelope peptide-cocktail

Infection of Indian-origin rhesus macaques by the simian human immunodeficiency virus (SHIV) is considered to be a suitable preclinical model for directly testing efficacy of vaccine candidates based on the HIV-1 envelope. We used this model for prophylactic vaccination with a peptide-cocktail comprised of highly conserved HIV-1 envelope sequences immunogenic/antigenic in macaques and humans. Separate groups of macaques were immunized with the peptide-cocktail by intravenous and subcutaneous routes using autologous dendritic cells (DC) and Freund's adjuvant, respectively. The vaccine elicited antigen specific IFN-gamma-producing cells and T-cell proliferation, but not HIV-neutralizing antibodies. The vaccinated animals also exhibited efficient cross-clade cytolytic activity against target cells expressing envelope proteins corresponding to HIV-1 strains representative of multiple clades that increased after intravenous challenge with pathogenic SHIV(KU2). Virus-neutralizing antibodies were either undetectable or present only transiently at low levels in the control as well as vaccinated monkeys after infection. Significant control of plasma viremia leading to undetectable levels was achieved in majority of vaccinated monkeys compared to mock-vaccinated controls. Monkeys vaccinated with the peptide-cocktail using autologous DC, compared to Freund's adjuvant, and the mock-vaccinated animals, showed significantly higher IFN-gamma production, higher levels of vaccine-specific IFN-gamma producing CD4(+) cells and significant control of plasma viremia. These results support DC-based vaccine delivery and the utility of the conserved HIV-1 envelope peptide-cocktail, capable of priming strong cell-mediated immunity, for potential inclusion in HIV vaccination strategies.     

Development of virus-specific immune responses in SHIV(KU)-infected macaques treated with PMPA

Therapeutic intervention with highly active antiretroviral therapy (HAART) can lead to the suppression of HIV viremia below the threshold of detection for several years. However, impact of HAART on reconstitution of virus-specific immune responses remains poorly understood. In this study, four macaques were infected with pathogenic SHIV(KU). One week postinoculation two of the four animals were treated with PMPA [9-R-(2-phosphophomethoxypropyl)adenine] daily for 83 days. Two other macaques, that did not receive treatment, exhibited explosive virus replication accompanied by a near total loss of CD4(+) T cells and succumbed to AIDS-related complications within 6 months of infection. These animals did not develop any virus-specific immune responses. On the contrary, the animals that received PMPA showed transient loss of CD4(+) T cells that recovered during the treatment period. The virus burden declined below the level of detection that rebounded soon after cessation of PMPA therapy. The virus replicated productively for several weeks before both animals controlled the productive replication of virus. This control of virus replication was found to be associated with the development of virus-specific neutralizing antibodies, T-helper cells, and CTLs. Although PMPA did not eliminate virus from the animals, it provided them with enough time to mount virus-specific immune responses that eventually controlled the virus replication in the blood. Our results suggest that antiretroviral therapy, if initiated early during infection, would help the host in mounting virus-specific immune responses that might control productive replication of the virus.     

中国恒河猴(Macaca mulatta)外周血Th17细胞及其在SHIV感染后的变化

目的: 对正常中国恒河猴(Macaca mulatta)及嵌合猿猴/人免疫缺陷病毒(SHIV)感染过的中国恒河猴外周血中CD4 IL-17A 和CD4-IL-17A T淋巴细胞的分布频率进行初步观察.方法: 用荧光染料标记的单克隆抗体(mAb)对10只中国恒河猴的外周血或PMA Ionomycin刺激后的PBMC细胞表面的CD3、 CD4、 CD8及细胞内IL-17A进行免疫荧光染色.用FACScalibaur获取染色后的样品, 所得数据用CellQuest进行分析.结果: 在6只正常中国恒河猴个体中均能检测到IL-17A 淋巴细胞, 未经刺激培养的样品中IL-17A 细胞频率很低, 但在短期刺激培养后淋巴细胞中具有约1.4%的淋巴细胞为IL-17A 细胞, 明显多于刺激前的IL-17A 淋巴细胞; 在短期刺激培养后CD3 CD4 淋巴细胞中大约有2.7%的IL-17A 细胞或CD4 Th17细胞.对4只SHIV感染个体中Th17细胞的初步分析未发现它们与正常个体间的统计学差异.结论: 与人类相似, 中国恒河猴的外周血中也存在一定比例的Th17细胞, 为进一步利用恒河猴AIDS动物模型分析HIV/AIDS与Th17细胞的关系提供了基础.     

Experimental Helicobacter pylori infection of rhesus macaques (Macaca mulatta)

The aim of the present study was to establish an animal model for Helicobacter pylori (H. pylori) infection at the German Primate Centre in rhesus monkeys (Macaca mulatta). During the experiments the susceptibility of three animals to different H. pylori strains of human origin was tested. In a follow-up study gastric biopsies from three different sites were investigated in regular intervals using microbiological, histological, electron microscopical and molecular biological methods to evaluate the presence of bacterial colonization and the occurrence of gastritis. It was possible to establish a persistent experimental infection. The rather long follow-up period of 18 months offered the possibility to demonstrate a permanent H. pylori infection in the gastric mucosa of the test animals. The three animals have now been successfully colonized with H. pylori for 18 months and presented a chronic active gastritis confirmed by microbiological and histological methods. By molecular typing, the identity of the isolates recovered from the animals was shown. It was possible to demonstrate that one infection strain outcompeted the second one. Taken together, prerequisites exist for making use of an attractive and useful animal model in rhesus monkeys especially for long term observations.     

Innate differences between simian-human immunodeficiency virus (SHIV)(KU-2)-infected rhesus and pig-tailed macaques in development of neurological disease

Murine gammaherpesvirus 68 replicates in the alveolar epithelium and induces an inflammatory infiltrate in the lung, following intranasal challenge, and is cleared 10 and 13 days after infection by a T-cell-dependent mechanism. In order to understand the development of the immune response to this virus and how leukocyte trafficking to the lung is regulated, chemokine expression during MHV-68 infection was examined in lung tissue using an RNase protection assay. Expression of RANTES, eotaxin, MIP-1 alpha, MIP-1 beta, IP-10, and MCP-1 was upregulated by day 7 after infection. Chemokine concentrations in lung lavage fluid were also determined by ELISA. MCP-1, RANTES, MIP-1 alpha, eotaxin, and KC were upregulated during MHV-68 infection. Most of these chemokines have been reported to be chemoattractants for either activated T cells or monocytes, which are the major cellular components of the inflammatory infiltrate induced by the virus. Upregulated expression of the corresponding receptors for the chemokines, including CCR1, CCR2, CCR3, CCR5, and CXCR3, coincided with the development of the inflammatory infiltrate. The chemokine levels peaked at around day 7 after infection, coinciding with peak viral titers and slightly preceding maximal T cell infiltration. In vitro chemotaxis assays confirmed that lung lavage fluid from MHV-68-infected mice had chemotactic activity, which was partially blocked by antibodies to IP-10 and RANTES. These observations suggest that the chemokines detected play an important role in regulating leukocyte trafficking to the lungs during MHV-68 infection.     

Selection for urease activity during Helicobacter pylori infection of rhesus macaques (Macaca mulatta)

Helicobacter pylori strain J166 recovered from experimentally inoculated rhesus monkeys had up to a 250-fold-increased urease activity over that before inoculation. This was found to result from the selection of urease positive J166 clones from a heterogeneous inoculum, which was predominantly urease negative due to a 1-bp insertion in the ureA gene. These results confirm the importance of urease for H. pylori colonization. Strain J166 is particularly well adapted to the rhesus monkey, since it colonized preferentially despite the fact that less than 0.1% of the inoculum was urease positive.     

Effect of 9-(S)-(2,3-dihydroxypropyl)adenine on experimental rabies infection in laboratory mice

9-(S)-(2,3-dihydroxypropyl)adenine (DHPA) exerted a pronounced influence on experimental rabies infection in laboratory mice. It was effective in a dose range from 1 to 100 mg/kg body weight. Intracerebral infection tended to be potentiated, intramuscular infection was partly or fully inhibited by the drug. Peroral administration was the most effective. The results of individual experiments varied and depended on the sensitivity of the virus strains employed, the dose of DHPA given and the time pattern of its administration.     

Nonpathogenic CCR2-tropic SIVrcm after serial passage and its effect on SIVmac infection of Indian rhesus macaques

The natural host of SIVrcm is the red-capped mangabey (Cercocebus torquatus torquatus). Although this virus infects macaques and human PBMCs, its pathogenic potential is unknown. We serially passaged SIVrcm through 9 rhesus macaques to assess its potential for virulence. SIVrcm infected all macaques with peak viremia 2 weeks postinfection yet viral loads decreased to undetectable levels about one month after inoculation. Remarkably, SIVrcm replication and virulence did not increase following 7 serial passages. While CD4+ T cells in the gut were decreased in early infection, proportions of memory CD4+CCR5+ T cells were not affected. Three SIVrcm-infected macaques were subsequently challenged with SIVmac251 to assess the potential for superinfection. Interestingly, animals previously infected with SIVrcm had 100 fold lower levels of SIVmac251 in plasma compared to naive animals inoculated with SIVmac251. These results suggest that SIVrcm is nonpathogenic and may be useful for examining effective immune responses in SIV infection.     

Differential effects of simian immunodeficiency virus infection on immune inductive and effector sites in the rectal mucosa of rhesus macaques

The rectal mucosa, a region involved in human immunodeficiency virus/simian immunodeficiency virus (SIV) infection and transmission, contains immune inductive sites, rectal lymphoid nodules (RLN), and effector sites, the lamina propria (LP). This study was designed to evaluate cell populations involved in rectal mucosal immune function in both RLN and LP, by immunocytochemical analysis of rectal mucosa from 11 SIV-infected (2 to 21 months postinfection) and five naive rhesus macaques. In the rectum, as previously observed in other intestinal regions, CD4(+) cells were dramatically reduced in the LP of SIV-infected macaques, but high numbers of CD4(+) cells remained in RLN indicating maintenance of T cell help in inductive sites. Cells expressing the mucosal homing receptor alpha4beta7 were dramatically decreased in the RLN and LP of most SIV-infected macaques. The RLN of both naive and SIV-infected macaques contained high numbers of CD68 + MHC-II+ macrophages and cells expressing the co-stimulatory molecules B7-2 and CD40, as well as IgM + MHCII+ and IgM + CD40+ B cells, indicating maintenance of antigen presentation capacity. The LP of all three macaques SIV-infected for 2 months contained many B7-2+ cells, suggesting increased activation of antigen-presenting cells. LP of SIV-infected rectal mucosa contained increased numbers of IgM+ cells, confirming previous observations in small intestine and colon. The data suggest that antigen-presentation capacity is maintained in inductive sites of SIV-infected rectal mucosa, but immune effector functions may be altered.     

Fatal immunopathogenesis by SIV/HIV-1 (SHIV) containing a variant form of the HIV-1SF33 env gene in juvenile and newborn rhesus macaques.

SIV/HIV-1 (SHIV) chimeric clones, constructed by substituting portions of the pathogenic molecular clone SIVmac239 with counterpart portions from HIV-1 clones, provide a means to analyze functions of selected HIV-1 genes in vivo in nonhuman primates. Our studies focused on SHIVSF33, which contains the vpu, tat, rev, and env genes of the cytopathic, T-cell line tropic clone HIV-1sf33 (subtype-B); this clone has a premature stop codon in the vpu gene. In three juvenile macaques inoculated intravenously with SHIVSF33, low-level persistent infection was established; no disease was observed for a period of >2 years. However, at approximately 16 months p.i., one of four SHIVSF33-infected juvenile macaques exhibited an increase in virus load, depletion of CD4(+) T cells in peripheral blood and lymph nodes, and other symptoms of simian AIDS (SAIDS). Virus recovered from this animal in the symptomatic stage was designated SHIVSF33a (A, adapted); this virus displayed multiple amino acid sequence changes throughout the HIV-1 env gene compared with the input SHIVSF33 clone. Additionally, a mutation in all clones from SHIVSF33a restored the open reading frame for the vpu gene. In vitro evaluations in tissue-culture systems revealed that SHIVSF33a replicated to higher levels and exhibited greater cytopathicity than SHIVSF33. Furthermore cloned env genes for SHIVSF33a were more fusogenic in a cell-fusion assay compared with the env gene of the SHIVSF33. Intravenous inoculation of SHIVsf33a into juvenile and newborn macaques resulted in a rapid decline in CD4(+) T cells to very low levels and development of a fatal AIDS-like disease. A cell-free preparation of this pathogenic chimeric virus also established persistent infection when applied to oral mucosal membranes of juvenile macaques and produced a fatal AIDS-like disease. These studies on pathogenic SHIVSF33a establish the basis for further investigations on the role of the HIV-1 env gene in virus adaptation and in mechanism(s) of immunodeficiency in primates; moreover, the chimeric virus SHIVSF33a can play a role in elucidating mucosal membrane transmission and development of antiviral vaccines in newborns as well as juvenile and adult macaques.     

Long-term effects of infant rearing condition on the acquisition of dominance rank in juvenile and adult rhesus macaques (Macaca mulatta)

We examined the effects of early rearing experience on the development of dominance status in 53 juvenile (age 3) and then in 38 adult (ages 5-8) rhesus macaques. Based on previous research investigating the behavioral outcomes of nursery-rearing, we predicted that mother-reared (MR) monkeys would outrank peer-only reared (PR) monkeys, which would in turn outrank surrogate/peer-reared (SPR) subjects. Juvenile MR and PR subjects did not differ in ranks, but monkeys from both rearing backgrounds outranked SPR cage-mates at age 3. Independent of rearing condition, high-ranking juveniles gained the most weight between ages 1-3, suggesting that low status may be associated with decreases in early weight gain. Adult MR subjects outranked both PR and SPR subjects, with PR animals occupying intermediate ranks. These results indicate that impoverished early experiences, such as adult absence and limited social interaction, are useful predictors of future social success in rhesus macaques.     

Antiallergic activity of 6-(2-cyclohexylethyl)-[1,3,4]thiadiazolo- [3,2-a]-1,2,3-triazolo-[4,5-d]pyrimidin-9(3H)-one (DS-4574) in rats.

The antiallergic activity of DS-4574 was evaluated in several commonly used rat models for allergic diseases. In passive cutaneous anaphylaxis, DS-4574 given intravenously and orally induced dose-dependent inhibition with ID50 values of 0.55 and 2.8 mg/kg, respectively. In contrast, this compound had no antagonistic activity against the histamine- and serotonin-induced cutaneous vascular permeability. In lung anaphylaxis, DS-4574 inhibited pulmonary function changes induced by the antigen in a dose-dependent manner when it was given intravenously and orally, the ID50 values being 0.04 and 0.89 mg/kg, respectively. DS-4574 also inhibited antigen-induced histamine and leukotriene release in passive peritoneal anaphylaxis following oral administration. In addition, this compound prevented antigen-induced histamine release in passively sensitized mast cells in vitro. These potent activities of DS-4574 in in vivo and in vitro models of immediate-type hypersensitivity reactions suggest that this compound could be useful in the treatment of allergic diseases including asthma.     

Novel application of nonhuman primate tethering system for evaluation of acute phase SIVmac251 infection in rhesus macaques (Macaca mulatta)

Infection of rhesus macaques with simian immunodeficiency virus (SIV) is the preferred animal model for the development and testing of human immunodeficiency virus (HIV) vaccines, and animals protected from SIV challenge by live attenuated vaccines are an invaluable tool for determining immune correlates of protection. The acute phase of SIV infection, in which immune responses are most critical for slowing disease progression, occurs within the first 4 weeks of exposure. The small window of time available for observing critical immune responses makes obtaining adequate blood samples with sufficient frequency difficult. This study is the first to apply a previously reported nonhuman primate (NHP) tether system to study viral immunology. The use of the tether allows for frequent blood sampling without using restraints or sedation, thereby reducing the potentially confounding physiological changes induced by stress. We performed comparative analysis of acute phase immune responses in vaccinated and unvaccinated animals challenged with SIV-mac251. Our results demonstrate live attenuated vaccine-induced protection, which is associated with small increases in the cytotoxic T-cell (CTL) response to immunodominant epitopes, but not with increases in antibody titers. Additionally, vaccination was shown to establish a pool of antigen-specific CD8+ memory cells available for expansion after challenge. The confirmatory nature of these data indicates the validity of using the tether system for evaluation of acute phase anti-SIV responses and can be applied to the study of immune responses in other viral infections in which frequent sampling in small windows of time would be useful.     

CD4+ lymphocytopenia in acute infection of Asian macaques by a vaginally transmissible subtype-C,CCR5-tropic Simian/Human Immunodeficiency Virus (SHIV)

An R5-tropic SHIV(CHN19P4) was previously generated using a primary HIV-1 subtype-C envelope. We have further characterized this SHIV in two species of macaques. To determine whether this isolate is transmissible vaginally, female pig-tailed macaques were inoculated with 2 x 10(3) TCID50 of SHIV(CHN19P4) by the vaginal route. Animals became infected with a high peak plasma viremia (>10(7) viral copies/mL) and rapid seroconversion. The viremia was accompanied by CD4+ lymphocytopenia in the gut lamina propria lymphocyte (LPL) population. Comparable CD4+ T-cell loss was not seen in peripheral blood and colonic lymph nodes. These findings demonstrate a unique R5-tropic SHIV that can be used to study envelope-related issues in vaginal transmission of the most prevalent subtype of HIV-1. We also found that rhesus macaques intravenously inoculated with 1 x 10(3) TCID50 of SHIV(CHN19P4) became infected and showed CD4+ lymphocytopenia in the gut LPL population. Despite inactivation of the vpu gene in SHIV(CHN19P4), the virus appears to target mainly gut-associated lymphoid tissues during the initial stage of infection as has been described for SHIV(SF162P), another R5-tropic (subtype B) recombinant virus. Our data indicate that the R5-mediated CD4+ lymphocytopenia in the gut is likely independent of HIV-1 genotypes and of the function of vpu at the acute phase of viral infection.     

Cationic ring opening polymerization of 4,5-dihydro-2-[2-(9-anthryl)ethyl]-1,3-oxazole

A new monomer, 4,5-dihydro-2-[2-(9-anthryl)ethyl]-1,3-oxazole ( 5 ) was synthesized and polymerized by cationic ring-opening isomerization. The polymerization was carried out in bulk or in solution, using methyl tosylate, ethylene ditosylate and α-tosyl-ω-tosyloxypoly(oxyethylene) [poly(ethylene oxide)ditosylate] as initiators. The polymers were characterized by IR, ¹H NMR and UV spectroscopy.     

Memory effects of arginine vasopressin (AVP) and [7-9] fragment of its peptide chain in rats

The effect of arginine vasopressin (AVP) and [7-9]-vasopressin on different memory types was examined in tests of active avoidance, passive avoidance and water maze after both intraventricular and subcutaneous injections. Results were compared with AVP and [7-9]AVP effects in open field and holeboard tests. Significant latency time elongation was observed after both icv and s.c. injections of AVP but not [7-9]AVP in the passive avoidance test. This effect was blocked by [beta-Mercapto-beta, beta-cyclopentanometyleno1-propionylo1-O-Et-Tyr2,Val4,Arg8]-vasopressin, an antagonist of V1 receptors. Injections of both peptides did not improve the performance in other performed tests including the open field test and the holeboard test. Therefore the results confirmed that AVP improves memory only in the passive avoidance test. This effect is probably mediated by central V1 receptors and is not induced by exploratory and locomotor activity impairment. Considering the above results together with the literature data, it seems that the most important fragment of AVP is [4-6] AVP.     

Steric effects on excimer formation in 2-amino-1,3-di(2-naphtyl)propane luminiscent markers covalently bonded to poly [N-(2-hydroxypropyl)methacrylamide]

The fluorescence spectra of poly[N-(2-hydroxypropyl)methacrylamide] (poly[1-(2-hydroxypropylaminocarbonyl)-1-methylethylene]) with covalently bonded luminiscent markers (2-amino-1, 3-di(2-naphthyl)propane (ADNP)) dissolved in methanol were measured over the temperature range 190–290 K. The effect of the spacer length on intramolecular excimer formation in ADNP was investigated. The ratio of the fluorescence intensities of the dimer I_D and the monomer I_M follows an Arrhenius temperature dependence in the temperature range 190–250 K with an activation energy independent of the spacer length. The numerical value of I_D/I_M at constant temperature increased with increasing spacer length reaching quickly a limiting value. The effect is explained by increasing hindrance to intramolecular excimer formation which is due—particularly for the shortest spacers—to increased steric interactions of the marker with the polymer chain.     

Effects of monotherapy with (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA) on the evolution of a primary Simian immunodeficiency virus (SIV) isolate

Determining the impact of antiretroviral therapy on virus evolution could advance the development of improved therapeutics/vaccines against HIV. Toward this goal, we analyzed virus burden, quasispecies complexity, and T cell responses in SIV/DeltaB670-infected rhesus macaques+/-treatment for 7 months with PMPA (2-30 weeks postinfection). Treatment divided the animals into two groups: poor responders (a reduction of < or =1 log) and responders (> or =2 log reduction) in virus burden. Virus evolution in poor responders and untreated controls was characterized by expression of a complex quasispecies that evolved as the disease progressed. This included the universal loss of a viral genotype selected against by in vitro passage in monkey cells and selected for by propagation in human cells. In contrast, a good response to PMPA was characterized by infection with a less complex quasispecies that evolved more slowly. Interestingly, in 2 of the best responders, the human-preferred genotype persisted until the study was discontinued (89 weeks p.i.). Neither virus burden nor the magnitude of the T cell response at 2 weeks postinfection predicted PMPA responsiveness. However, responders expressed a less complex quasispecies than nonresponders prior to treatment. These data suggest a role for intrinsic host factors in treatment responsiveness, and lend support for therapeutic vaccination as an adjunct to effective therapy.