An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.     

恒河猴Mamu-A^＊01基因与SIV／SHIV感染相关的研究进展

SIV／SHIV感染的恒河猴是研究艾滋病及艾滋病药物筛选、疫苗评价较理想的动物模型。MHC在细胞免疫中起着关键作用,研究表明,MHC-I类分子的多态性与SIV／SHIV感染者的疾病进展有着明显的关联作用,Mamu-A^＊01是恒河猴中的一种MHC-I类分子,它可以呈递特定的病毒蛋白片段到细胞的表面,从而激发CTL反应。国外发现Mamu-A^＊01阳性的猴艾滋病恒河猴会出现疾病进展缓慢,存活时间长等特征。本文就恒河猴Mamu-A^＊01基因与SIV／SHIV感染相关的研究进展做一综述,以期进一步加深对MHC在疫苗研究中的作用的了解,并促进更行之有效地对HIV／AIDS疫苗进行评价。     

Acute Effects of Pathogenic Simian-Human Immunodeficiency Virus Challenge on Vaccine-Induced Cellular and Humoral Immune Responses to Gag in Rhesus Macaques

Simian-human immunodeficiency virus (SHIV) infection in macaques provides a convenient model for testing vaccine efficacy and for understanding viral pathogenesis in AIDS. We immunized macaques with recombinant, Salmonella typhimurium (expressing Gag) or soluble Gag in adjuvant to generate T-cell-dependent lymphoproliferative or serum antibody responses. Immunized animals were challenged by intrarectal inoculation with SHIV89.6PD. Virus infection was accompanied by rapid losses of lymphoproliferative responses to Gag or phytohemagglutinin. By 8 weeks, mitogen responses recovered to near normal levels but antigen-specific immunity remained at low or undetectable levels. Serum antibody levels were elevated initially by virus exposure but soon dropped well below levels achieved by immunization. Our studies show a rapid depletion of preexisting Gag-specific CD4⁺ T cells that prevent or limit subsequent antiviral cellular and humoral immune responses during acute SHIV infection.     

Temporal Analyses of Virus Replication, Immune Responses, and Efficacy in Rhesus Macaques Immunized with a Live, Attenuated Simian Immunodeficiency Virus Vaccine

Despite evidence that live, attenuated simian immunodeficiency virus (SIV) vaccines can elicit potent protection against pathogenic SIV infection, detailed information on the replication kinetics of attenuated SIV in vivo is lacking. In this study, we measured SIV RNA in the plasma of 16 adult rhesus macaques immunized with a live, attenuated strain of SIV (SIVmac239Δnef). To evaluate the relationship between replication of the vaccine virus and the onset of protection, four animals per group were challenged with pathogenic SIVmac251 at either 5, 10, 15, or 25 weeks after immunization. SIVmac239Δnef replicated efficiently in the immunized macaques in the first few weeks after inoculation. SIV RNA was detected in the plasma of all animals by day 7 after inoculation, and peak levels of viremia (10⁵ to 10⁷ RNA copies/ml) occurred by 7 to 12 days. Following challenge, SIVmac251 was detected in all of the four animals challenged at 5 weeks, in two of four challenged at 10 weeks, in none of four challenged at 15 weeks, and one of four challenged at 25 weeks. One animal immunized with SIVmac239Δnef and challenged at 10 weeks had evidence of disease progression in the absence of detectable SIVmac251. Although complete protection was not achieved at 5 weeks, a transient reduction in viremia (approximately 100-fold) occurred in the immunized macaques early after challenge compared to the nonimmunized controls. Two weeks after challenge, SIV RNA was also reduced in the lymph nodes of all immunized macaques compared with control animals. Taken together, these results indicate that host responses capable of reducing the viral load in plasma and lymph nodes were induced as early as 5 weeks after immunization with SIVmac239Δnef, while more potent protection developed between 10 and 15 weeks. In further experiments, we found that resistance to SIVmac251 infection did not correlate with the presence of antibodies to SIV gp130 and p27 antigens and was achieved in the absence of significant neutralizing activity against the primary SIVmac251 challenge stock.     

Viral RNA Levels and env Variants in Semen and Tissues of Mature Male Rhesus Macaques Infected with SIV by Penile Inoculation

HIV is shed in semen but the anatomic site of virus entry into the genital secretions is unknown. We determined viral RNA (vRNA) levels and the envelope gene sequence in the SIVmac 251 viral populations in the genital tract and semen of 5 adult male rhesus monkeys (Macaca mulatta) that were infected after experimental penile SIV infection. Paired blood and semen samples were collected from 1–9 weeks after infection and the monkeys were necropsied eleven weeks after infection. The axillary lymph nodes, testes, epididymis, prostate, and seminal vesicles were collected and vRNA levels and single-genome analysis of the SIVmac251 env variants was performed. At the time of semen collection, blood vRNA levels were between 3.09 and 7.85 log10 vRNA copies/ml plasma. SIV RNA was found in the axillary lymph nodes of all five monkeys and in 3 of 5 monkeys, all tissues examined were vRNA positive. In these 3 monkeys, vRNA levels (log10 SIVgag copies/ug of total tissue RNA) in the axillary lymph node (6.48±0.50) were significantly higher than in the genital tract tissues: testis (3.67±2.16; p<0.05), epididymis (3.08±1.19; p<0.0001), prostate (3.36±1.30; p<0.01), and seminal vesicle (2.67±1.50; p<0.0001). Comparison of the SIVmac251 env viral populations in blood plasma, systemic lymph node, and genital tract tissues was performed in two of the macaques. Visual inspection of the Neighbor-Joining phylograms revealed that in both animals, all the sequences were generally distributed evenly among all tissue compartments. Importantly, viral populations in the genital tissues were not distinct from those in the systemic tissues. Our findings demonstrate striking similarity in the viral populations in the blood and male genital tract tissues within 3 months of penile SIV transmission.     

Evolutionary Distance of Amino Acid Sequence Orthologs across Macaque Subspecies: Identifying Candidate Genes for SIV Resistance in Chinese Rhesus Macaques

We use the Reciprocal Smallest Distance (RSD) algorithm to identify amino acid sequence orthologs in the Chinese and Indian rhesus macaque draft sequences and estimate the evolutionary distance between such orthologs. We then use GOanna to map gene function annotations and human gene identifiers to the rhesus macaque amino acid sequences. We conclude methodologically by cross-tabulating a list of amino acid orthologs with large divergence scores with a list of genes known to be involved in SIV or HIV pathogenesis. We find that many of the amino acid sequences with large evolutionary divergence scores, as calculated by the RSD algorithm, have been shown to be related to HIV pathogenesis in previous laboratory studies. Four of the strongest candidate genes for SIV_mac resistance in Chinese rhesus macaques identified in this study are CDK9, CXCL12, TRIM21, and TRIM32. Additionally, ANKRD30A, CTSZ, GORASP2, GTF2H1, IL13RA1, MUC16, NMDAR1, Notch1, NT5M, PDCD5, RAD50, and TM9SF2 were identified as possible candidates, among others. We failed to find many laboratory experiments contrasting the effects of Indian and Chinese orthologs at these sites on SIV_mac pathogenesis, but future comparative studies might hold fertile ground for research into the biological mechanisms underlying innate resistance to SIV_mac in Chinese rhesus macaques.     

预防性给予维生素D对实验性脑疟前炎症应答的影响

通过预防性给予维生素D(Vitamin D,VD)治疗,研究VD对实验性脑疟(ECM)模型鼠感染结局及免疫应答的影响,进一步阐明VD的免疫调节作用。通过P.b ANKA感染的C57BL/6小鼠建立ECM模型,通过ELISA和Griess法检测了预防给予VD对实验组小鼠脾上清和血清中前炎性细胞因子(IFN-γ、TNF-α和NO)和抗炎性细胞因子(IL-10)水平的影响,并检测了血脑屏障通透性的变化。结果显示,VD处理能够显著延长宿主生存期,同时显著降低血清和脾上清中的前炎性细胞因子水平,显著提升抗炎性细胞因子水平,从而保护血脑屏障完整性,进而抑制脑疟发生。结果表明,预防性给予VD治疗可以通过抑制宿主前炎症免疫应答以防止脑疟发生。     

Plasmodium vivax VIR Proteins Are Targets of Naturally-Acquired Antibody and T Cell Immune Responses to Malaria in Pregnant Women

P. vivax infection during pregnancy has been associated with poor outcomes such as anemia, low birth weight and congenital malaria, thus representing an important global health problem. However, no vaccine is currently available for its prevention. Vir genes were the first putative virulent factors associated with P. vivax infections, yet very few studies have examined their potential role as targets of immunity. We investigated the immunogenic properties of five VIR proteins and two long synthetic peptides containing conserved VIR sequences (PvLP1 and PvLP2) in the context of the PregVax cohort study including women from five malaria endemic countries: Brazil, Colombia, Guatemala, India and Papua New Guinea (PNG) at different timepoints during and after pregnancy. Antibody responses against all antigens were detected in all populations, with PNG women presenting the highest levels overall. P. vivax infection at sample collection time was positively associated with antibody levels against PvLP1 (fold-increase: 1.60 at recruitment -first antenatal visit-) and PvLP2 (fold-increase: 1.63 at delivery), and P. falciparum co-infection was found to increase those responses (for PvLP1 at recruitment, fold-increase: 2.25). Levels of IgG against two VIR proteins at delivery were associated with higher birth weight (27 g increase per duplicating antibody levels, p<0.05). Peripheral blood mononuclear cells from PNG uninfected pregnant women had significantly higher antigen-specific IFN-γ T_H1 responses (p=0.006) and secreted less pro-inflammatory cytokines TNF and IL-6 after PvLP2 stimulation than P. vivax-infected women (p<0.05). These data demonstrate that VIR antigens induce the natural acquisition of antibody and T cell memory responses that might be important in immunity to P. vivax during pregnancy in very diverse geographical settings.     

丙型肝炎病毒多表位抗原在恒河猴中的免疫应答及保护性试验

利用HCV抗原多表位来研制HCV疫苗是目前的一个新方向.本研究利用HCV的HCV的5个保守表位串联,并加入破伤风类毒素上的一个T细胞激活位点,设计成一个HCV多表位抗原基因PCX,在大肠杆菌中表达,用此蛋白免疫恒河猴,诱导猴体产生了较高的抗体水平,滴度达11000以上,在免疫后的60周抗体滴度仍达140以上.同时,在免疫后6周用人HCV阳性血清攻击猴子,免疫PCX的猴子出现一过性ALT升高,在攻击后三周内用RT-PCR检测到猴血清内HCV的RNA阳性.结果表明,免疫多表位的PCX蛋白可以诱导机体产生高水平的免疫应答.     

丙型肝炎病毒多表位抗原在恒河猴中的免疫应答及保护性试验

利用ＨＣＶ抗原多表位来研制ＨＣＶ疫苗是目前的一个新方向。本研　究利用ＨＣＶ的ＨＣＶ的５个保守表位串联,并加入破伤风类毒素上的一个Ｔ细胞激活位点,设计成　一个ＨＣＶ多表位抗原基因ＰＣＸ,在大肠杆菌中表达,用此蛋白免疫恒河猴,诱导猴体产生了较　高的抗体水平,滴度达１∶１０００以上,在免疫后的６０周抗体滴度仍达１∶４０以上。同时,在免　疫后６周用人ＨＣＶ阳性血清攻击猴子,免疫ＰＣＸ的猴子出现一过性ＡＬＴ升高,在攻击后三周内用　ＲＴ－ＰＣＲ检测到猴血清内ＨＣＶ的ＲＮＡ阳性。结果表明,免疫多表位的ＰＣＸ蛋白可以诱导机体产生　高水平的免疫应答。     

Maladjusted Host Immune Responses Induce Experimental Cerebral Malaria-Like Pathology in a Murine Borrelia and Plasmodium Co-Infection Model

In the Plasmodium infected host, a balance between pro- and anti-inflammatory responses is required to clear the parasites without inducing major host pathology. Clinical reports suggest that bacterial infection in conjunction with malaria aggravates disease and raises both mortality and morbidity in these patients. In this study, we investigated the immune responses in BALB/c mice, co-infected with Plasmodium berghei NK65 parasites and the relapsing fever bacterium Borrelia duttonii. In contrast to single infections, we identified in the co-infected mice a reduction of L-Arginine levels in the serum. It indicated diminished bioavailability of NO, which argued for a dysfunctional endothelium. Consistent with this, we observed increased sequestration of CD8+ cells in the brain as well over expression of ICAM-1 and VCAM by brain endothelial cells. Co-infected mice further showed an increased inflammatory response through IL-1β and TNF-α, as well as inability to down regulate the same through IL-10. In addition we found loss of synchronicity of pro- and anti-inflammatory signals seen in dendritic cells and macrophages, as well as increased numbers of regulatory T-cells. Our study shows that a situation mimicking experimental cerebral malaria (ECM) is induced in co-infected mice due to loss of timing and control over regulatory mechanisms in antigen presenting cells.     

以恒河猴为模型的DNA疫苗的免疫保护作用研究

研究了以霍乱毒素B亚基（CTB）为载体的重组疟疾多价抗原（AWTE）表位的DNA疫苗在恒河猴中的免疫原性及对相应疟原虫感染的免疫保护作用。结果表明DNA疫苗组免疫2次后即产生了较高水平的细胞免疫和体液免疫,免疫后91天用1.25×10     

Model for Concomitant Microdialysis Sampling of the Pons and Cerebral Cortex in Rhesus Macaques (Macaca mulatta)

Pediatric diffuse intrinsic pontine gliomas are aggressive brainstem tumors that fail to respond to treatment. We hypothesize that the protective features of the pons may hinder chemotherapeutic agents from entering pontine tissue compared with cortical brain tissue. To test this hypothesis, we developed a unique nonhuman primate model using microdialysis, a continuous in vivo extracellular sampling technique, to compare drug exposure concurrently in pontine tissue, cortical tissue, CSF, and plasma after intravenous administration of chemotherapeutic agents. The surgical coordinates and approach for microdialysis cannula–probe placement were determined in 5 adult male rhesus monkeys (Macaca mulatta) by using MRI. Microdialysis cannulas–probes were implanted stereotactically in the brain, retrodialysis was performed to measure relative recovery, and a 1-h intravenous infusion of temozolomide was administered. Continuous microdialysis samples were collected from the pons and cortex over 4 h with concurrent serial plasma and CSF samples. Postsurgical verification of microdialysis cannula–probe placement was obtained via MRI in 3 macaques and by gross pathology in all 5 animals. The MRI-determined coordinates and surgical methodologies resulted in accurate microdialysis probe placement in the pons and cortex in 4 of the 5 macaques. Histologic examination from these 4 animals revealed negligible tissue damage to the pontine and cortical tissue from microdialysis. One macaque was maintained for 8 wk and had no deficits attributed to the procedure. This animal model allows for the determination of differences in CNS penetration of chemotherapeutic agents in the pons, cortex, and CSF after systemic drug administration.Abbreviations: DIPG, diffuse intrinsic pontine glioma; ECF, extracellular fluidPediatric diffuse intrinsic pontine gliomas (DIPG) are aggressive tumors that cannot be surgically resected due to their location, and are resistant to chemotherapeutic and radiation therapies. As a result, children with DIPG have a dismal prognosis with median survival less than one year from diagnosis. One hypothesis for the poor efficacy of treatment is that innate CNS protective features, such as the blood–brain barrier and the blood–CSF barrier, shield the brainstem to a higher degree given its critical functions, and isolate pontine gliomas from treatment. To test this hypothesis, we developed a nonhuman primate model in rhesus monkeys to evaluate pontine tissue pharmacokinetics by using microdialysis, a continuous in vivo extracellular sampling technique based on diffusion. Microdialysis is the ‘gold standard’ for in vivo sampling methodologies in the CNS, enabling the collection of extracellular tissue fluid via passive diffusion by using a semipermeable membrane probe.A nonhuman primate model demonstrating the feasibility of microdialysis sampling from cortical brain tissue with concurrent pharmacokinetic sampling during chemotherapeutic drug administration has previously been established,^3-5,7 but there are no current animal models that measure drug penetration into the pons. The location of the pons deep within the brain, as well as the vital brainstem functions associated with the pons, present additional obstacles to accurate microdialysis probe placement and sample collection. The objectives of the current study were to develop imaging and surgical procedures for the accurate placement of a microdialysis probe within the pons of rhesus monkeys for sample collection, to establish a method to perform microdialysis simultaneously in multiple CNS regions, and to develop a mechanism to perform repeated microdialysis in the same areas with a single invasive surgical procedure. This model allows for the pharmacokinetic comparison of drug penetration into pontine tissue, in conjunction with cortical tissue, plasma, and CSF, after intravenous administration.     

Role of the SH3-Ligand Domain of Simian Immunodeficiency Virus Nef in Interaction with Nef-Associated Kinase and Simian AIDS in Rhesus Macaques

The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) is dispensable for viral replication in T-cell lines; however, it is essential for high virus loads and progression to simian AIDS (SAIDS) in SIV-infected adult rhesus macaques. Nef proteins from HIV type 1 (HIV-1), HIV-2, and SIV contain a proline-Xaa-Xaa-proline (PxxP) motif. The region of Nef with this motif is similar to the Src homology region 3 (SH3) ligand domain found in many cell signaling proteins. In virus-infected lymphoid cells, Nef interacts with a cellular serine/threonine kinase, designated Nef-associated kinase (NAK). In this study, analysis of viral clones containing point mutations in the nef gene of the pathogenic clone SIVmac239 revealed that several strictly conserved residues in the PxxP region were essential for Nef-NAK interaction. The results of this analysis of Nef mutations in in vitro kinase assays indicated that the PxxP region in SIV Nef was strikingly similar to the consensus sequence for SH3 ligand domains possessing the minus orientation. To test the significance of the PxxP motif of Nef for viral pathogenesis, each proline was mutated to an alanine to produce the viral clone SIVmac239-P¹⁰⁴A/P¹⁰⁷A. This clone, expressing Nef that does not associate with NAK, was inoculated into seven juvenile rhesus macaques. In vitro kinase assays were performed on virus recovered from each animal; the ability of Nef to associate with NAK was restored in five of these animals as early as 8 weeks after infection. Analysis of nef genes from these viruses revealed patterns of genotypic reversion in the mutated PxxP motif. These revertant genotypes, which included a second-site suppressor mutation, restored the ability of Nef to interact with NAK. Additionally, the proportion of revertant viruses increased progressively during the course of infection in these animals, and two of these animals developed fatal SAIDS. Taken together, these results demonstrated that in vivo selection for the ability of SIV Nef to associate with NAK was correlated with the induction of SAIDS. Accordingly, these studies implicate a role for the conserved SH3 ligand domain for Nef function in virally induced immunodeficiency.