ELISPOT方法检测SIVmac239感染猴特异性细胞免疫水平

目的为了完善现有的SIV／恒河猴模型,掌握恒河猴被SIV感染后体内细胞免疫应答状态,为评价HIV疫苗提供方法和数据上的参考,我们测定了SIV感染猴体内病毒特异性的细胞免疫水平。方法实验前选出4只无SIV、sTLV、SRV／D和B病毒感染的恒河猴,用SIVmac239病毒液静脉感染实验猴,使用RT-PCR、流氏细胞术和ELISPOT等方法,监测SIVmac239病毒在恒河猴体内复制情况、感染猴的外周免疫损伤情况和细胞免疫情况,持续测定一年。结果实验结果显示IFN-γ ELISPOT方法能有效的评估实验猴的细胞免疫情况,IFN—YELISPOT结果和CD4＋T细胞数无相关性,与血浆病毒载量稍有相关。结论本实验明确了SIVmac239感染中国恒河猴体内CTL的基本趋势和范围,了解了外周血病毒载量、外周免疫损伤与细胞免疫状况之间的联系,完善了SIV／SAIDS模型评价指标,为使用此模型评价抗病毒药物或疫苗提供了基础条件。     

猴免疫缺陷病毒（SIV）感染恒河猴血液学和血液生化的动态变化

目的探讨SIVmac239感染中国恒河猴的血液学和血清生物化学的变化,研究艾滋病的发生机制。方法 20只恒河猴感染SIVmac239后分别在感染前和感染后2、6、9、12、15和18个月在猴空腹状态用盐酸氯胺酮麻醉下静脉釆血。抗凝血用全自动血液细胞分析仪检测血液常规,血清用全自动生化分析仪检测生化指标。结果SIV感染猴红白细胞计数呈逐渐的减少。淋巴细胞计数有较明显减/增的波动。血清酶类ALT和AST没有明显的上升,但在15月时均有所下降P〈0.05～P〈0.01。LDH持续降低,原因未明。CK则逐月上升,至12月时达高峰,后又有所下降。血尿素氮逐月上升,并在6～18个月时P〈0.01。总蛋白量上升,白蛋白逐月降低,球蛋白升高,白蛋白/球蛋白比率倒置。SIV感染猴2个月后总胆固醇、甘油三脂、低密度脂蛋白和高密度脂蛋白均呈上升,至12月时略有下降,但到18月又上升。血糖（GLU）在SIV感染后逐月上升,后期上升更为明显,这可能与胰岛的损害有关。结论 SIVmac239感染猴后,通过SIV侵入相关的器官病变和体液调节失衡导致血液学和血液生物化学产生异常,从而提供有关猴艾滋病的一些基础资料,这将对研究人的艾滋病也许有所帮助。     

恒河猴感染SARS-CoV的病毒学、血清学检测

目的对感染SARS-CoV的8只恒河猴进行病毒学、血清学指标检测。方法SARS-CoV经鼻腔接种8只恒河猴,在感染的第1天开始到5、7、10、15、20、30和60天分别安乐处死时,不同时间取咽拭子、血液和脏器,进行病毒分离,RT-PCR检测和抗体测定。结果RT-PCR证实感染病毒检出时间为5~16d,8只猴中的5只分离到了病毒,感染15d后可检测到抗体。结论感染SARS-CoV的恒河猴不仅出现与SARS患者类似的临床和病理学改变,也在一定时期内排毒,出现特异免疫反应,这些指标均可作为药物筛选、疫苗评价等方面的重要参数。     

SHIV-KB9感染中国恒河猴动物模型的建立

目的为了进一步确证SHIV-KB9感染中国恒河猴的病毒浓度范围,测试动物对病毒的适应性,明确该动物模型的可重复性。方法实验前采集猴血清并进行血清学检查。选出4只无SIV、STLV、SRV/D和B病毒感染的恒河猴,分别用10倍系列稀释的病毒液静脉感染实验猴,使用流氏细胞术、血常规、病毒分离、DNA-PCR和RT-PCR等方法确定实验猴是否被感染,以及感染后恒河猴体内病毒复制和免疫细胞损伤情况。结果实验猴的血浆病毒载量、病毒分离结果、CD4＋/CD8＋比值和CD4＋T细胞数等证实,4.8×105 copies/mL以上浓度的SHIV-KB9病毒液能成功感染中国恒河猴。结论本研究进一步明确了SHIV-KB9感染中国恒河猴的有效病毒浓度范围,确定了SHIV-KB9病毒感染中国恒河猴的病毒学、免疫学的测定指标,成功的建立了SHIV-KB9/中国恒河猴动物模型。     

利用恒河婴猴模型对肠道病毒71型传播感染特征的生物学分析

本文在前期工作基础上,进一步对肠道病毒71型(EV71)从恒河婴猴的感染个体向其他未感染个体传播的可能性及相关生物学特性做了初步分析.通过喷雾形式经呼吸道感染1~2月龄恒河婴猴(A组);在观察临床症状同时,于感染后第7天,取该组动物粪便处理后,将上清液以喷雾形式经呼吸道感染新的婴猴个体(B组),随后对该次代感染个体进行...     

B亚型SHIV_（SF162p3）中国恒河猴小剂量多次阴道黏膜感染

目的模拟HIV性传播感染特点进行中国恒河猴阴道黏膜小剂量多次感染研究,为我国艾滋病疫苗有效性评价提供新的模型构建思路。方法选用20-30TCID50剂量的SHIVSF162p3病毒阴道黏膜途径感染六只成年雌性中国恒河猴,共感染13次,每次攻毒间隔4～7 d。采取测定血浆病毒载量和外周血CD4＋∶CD8＋。结果 6只中国恒河猴经13次病毒攻击后,经检测均建立系统性感染,血浆病毒载量呈阳性;CD4＋∶CD8＋均有下降。结论成功建立了中国恒河猴阴道黏膜小剂量多次感染模型,为艾滋病研究提供了新的更接近于自然感染状态的模型建立模式。     

表现神经症状的SIVmac251感染猴大脑基底节病毒gp120序列变异分析

目的 研究猴免疫缺陷病毒SIVmac251在中国恒河猴感染传代过程中产生的可能的神经侵袭性和神经嗜性及其分子机制.方法 从静脉感染SIVmac251-155p6N的8只实验猴中出现严重神经症状的1只猴中,监测病毒及免疫指标变化,观察临床症状、猴脑组织病变,单拷贝PCR扩增病毒gp120序列并分析变异及糖基化位点变化情况.结果 感染猴晚期出现明显艾滋病脑病症状,病理切片显示脑组织出现多核巨细胞及神经元变性、坏死.脑基底节分离出单一序列病毒,其氨基酸序列与血浆病毒及感染毒株SlVmac251-155p6序列差异主要位于Gp120的V1和V4区,并且在C1区66位出现一个糖基化位点缺失.结论 SIVmac251在猴体长期传代过程中表现出神经嗜性毒株的特征,对AIDS脑病研究具有重要意义.     

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。     

Establishment of AIDS Animal Model with SIVmac239 Infected Chinese Rhesus Monkey

In the present research,two Chinese rhesus monkeys were inoculated intravenously with 5000 TCID50 of SIVmac239. The changes in the numbers of CD4 T lymphocyte in peripheral blood,plasma viral loads,proviral DNA and humoral antibodies against virus were periodically monitored during 121 days. At the early stage of infection,proviral DNA had been detected in PBMCs,and infectious SIVmac239 virus had been isolated from PBMCs. At the same period,the numbers of CD4 T lymphocytes were significantly decreased,and maintained at low level during the 121-day period of infection. Plasma viral loads reached the peak at week 2 post-inoculation and kept at a steady state subsequently. Moreover,antibodies against viral proteins were detected from plasma. All the results showed that the two Chinese rhesus monkeys had been infected with SIVmac239 successfully. This animal model can be applied for further AIDS researches.     

Diverse Host Responses and Outcomes following Simian Immunodeficiency Virus SIVmac239 Infection in Sooty Mangabeys and Rhesus Macaques

Sooty mangabeys naturally infected with simian immunodeficiency virus (SIV) do not develop immunodeficiency despite the presence of viral loads of 10⁵ to 10⁷ RNA copies/ml. To investigate the basis of apathogenic SIV infection in sooty mangabeys, three sooty mangabeys and three rhesus macaques were inoculated intravenously with SIVmac239 and evaluated longitudinally for 1 year. SIVmac239 infection of sooty mangabeys resulted in 2- to 4-log-lower viral loads than in macaques and did not reproduce the high viral loads observed in natural SIVsmm infection. During acute SIV infection, polyclonal cytotoxic T-lymphocyte (CTL) activity coincident with decline in peak plasma viremia was observed in both macaques and mangabeys; 8 to 20 weeks later, CTL activity declined in the macaques but was sustained and broadly directed in the mangabeys. Neutralizing antibodies to SIVmac239 were detected in the macaques but not the mangabeys. Differences in expression of CD38 on CD8⁺ T lymphocytes or in the percentage of naive phenotype T cells expressing CD45RA and CD62L-selection did not correlate with development of AIDS in rhesus macaques. In macaques, the proportion of CD4⁺ T lymphocytes expressing CD25 declined during SIV infection, while in mangabeys, CD25-expressing CD4⁺ T lymphocytes increased. Longitudinal evaluation of cytokine secretion by flow cytometric analysis of unstimulated lymphocytes revealed elevation of interleukin-2 and gamma interferon in a macaque and only interleukin-10 in a concurrently infected mangabey during acute SIV infection. Differences in host responses following experimental SIVmac239 infection may be associated with the divergent outcome in sooty mangabeys and rhesus macaques.     

Establishment of AIDS animal model with SIVmac239 infected Chinese rhesus monkey

In the present research, two Chinese rhesus monkeys were inoculated intravenously with 5000 TCID₅₀ of SIVmac239. The changes in the numbers of CD4⁺ T lymphocyte in peripheral blood, plasma viral loads, proviral DNA and humoral antibodies against virus were periodically monitored during 121 days. At the early stage of infection, proviral DNA had been detected in PBMCs, and infectious SIVmac239 virus had been isolated from PBMCs. At the same period, the numbers of CD4⁺ T lymphocytes were significantly decreased, and maintained at low level during the 121-day period of infection. Plasma viral loads reached the peak at week 2 post-inoculation and kept at a steady state subsequently. Moreover, antibodies against viral proteins were detected from plasma. All the results showed that the two Chinese rhesus monkeys had been infected with SIVmac239 successfully. This animal model can be applied for further AIDS researches. These authors contributed equally to this work.     

Persistent Infection of Rhesus Macaques by the Rev-Independent Nef(−) Simian Immunodeficiency Virus SIVmac239: Replication Kinetics and Genomic Stability

We generated previously a Nef(-), replication-competent clone of SIVmac239 in which the Rev protein and the Rev-responsive element were replaced by the constitutive transport element (CTE) of simian retrovirus type 1 (A. S. von Gegerfelt and B. K. Felber, Virology 232:291-299, 1997). In the present report, we show that this virus was able to infect and replicate in rhesus macaques. The Rev-independent Nef(-) simian immunodeficiency virus induced a persistent humoral immune response in all monkeys, although viral loads were very low. Upon propagation in the monkeys, the genotype remained stable and the virus retained its in vitro growth characteristics. The infected monkeys showed normal hematological values and no signs of disease at more than 18 months post-virus exposure. Therefore, replacement of the essential Rev regulation by the CTE generated a virus variant that retained its replicative capacity both in vitro and in vivo, albeit at low levels.     

Thenef gene of SIVmac239 is necessary for efficient growth in H9 cells

AIDS viruses require an intact functionalnef gene in order to inducedisease. The nonpathogenic molecular cloned virus SIVmac239nef-deletion encodes a truncatednef gene. This attenuated reading frame is expressed both in vitro and in a virus-infected animal in vivo. Encoding the first 58 amino acids of Nef, the reading frame retained its ability to down-modulate CD4 from the surface of T cells. CD4-down-modulated stable cell lines expressing full-length and truncatednef genes were significantly less infected by SIV. SIV-mac239nef-open and SIVmacnef-deletion encoding a truncatednef clearly differed in replication kinetics in H9 cells and H9-derived cell lines. SIV-mac239nef-deletion replication was delayed in H9.     

Recombinant Yellow Fever Vaccine Virus 17D Expressing Simian Immunodeficiency Virus SIVmac239 Gag Induces SIV-Specific CD8+ T-Cell Responses in Rhesus Macaques

Here we describe a novel vaccine vector for expressing human immunodeficiency virus (HIV) antigens. We show that recombinant attenuated yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 Gag sequences can be used as a vector to generate SIV-specific CD8⁺ T-cell responses in the rhesus macaque. Priming with recombinant BCG expressing SIV antigens increased the frequency of these SIV-specific CD8⁺ T-cell responses after recombinant YF17D boosting. These recombinant YF17D-induced SIV-specific CD8⁺ T cells secreted several cytokines, were largely effector memory T cells, and suppressed viral replication in CD4⁺ T cells.None of the vaccine regimens tested in human immunodeficiency virus (HIV) vaccine efficacy trials to date have either reduced the rate of HIV infection or reduced the level of HIV replication. Structural features and the enormous variability of the envelope glycoprotein have frustrated efforts to induce broadly reactive neutralizing antibodies against HIV (10). Investigators have therefore focused their attention on T-cell-based vaccines (40). Simian immunodeficiency virus (SIV) challenge of rhesus macaques vaccinated with T-cell-based vaccines has shown that it is possible to control virus replication after SIV infection (22, 41, 42). The recent STEP trial of a recombinant Ad5-vectored vaccine was widely seen as an important test of this concept (http://www.hvtn.org/media/pr/step111307.html) (9, 25). Unfortunately, vaccinees became infected at higher rates than the controls (9). While it is still not clear what caused the enhanced infection rate in the vaccinated group, future Ad5-based human vaccine trials may be difficult to justify. We therefore need to develop new vaccine vectors for delivering SIV and HIV genes. Several other viral vectors currently under consideration include nonreplicating adenovirus (Ad)-based vectors (1, 21, 22), Venezuelan equine encephalitis (VEE) virus (12, 20), adeno-associated virus (AAV) (19), modified vaccinia virus Ankara (MVA) (3, 4, 13, 15, 18, 38), NYVAC (6), cytomegalovirus (CMV) (16), and replicating Ad (30). However, only a few of these have shown promise in monkey trials using rigorous SIV challenges.We explored whether the small (11-kb) yellow fever vaccine flavivirus 17D (YF17D) might be a suitable vector for HIV vaccines. The YF17D vaccine is inexpensive, production and quality control protocols already exist, and it disseminates widely in vivo after a single dose (27). Importantly, methods for the manipulation of the YF17D genome were recently established (7, 8, 24, 28). This effective vaccine has been safely used on >400 million people in the last 70 years (27). Additionally, the YF17D strain elicits robust CD8⁺ T-cell responses in humans (26). Chimeric YF17D is presently being developed as a vaccine for other flaviviruses, such as Japanese encephalitis virus (28), dengue virus (14), and West Nile virus (29). Inserts expressing a malaria B-cell epitope have been engineered into the E protein of YF17D (7). In murine models, recombinant YF17D viruses have generated robust and specific responses to engineered antigens inserted between the 2B and NS3 proteins in vivo (24, 35).We first used the YF17D vaccine virus to infect four Mamu-A*01-positive macaques. The vaccine virus replicated in these four animals and induced neutralizing antibodies in all four macaques by 2 weeks postvaccination (Fig. 1A and B). To monitor the CD8⁺ T-cell immune response against YF17D, we scanned its proteome for peptides that might bind to Mamu-A*01 using the major histocompatibility complex (MHC) pathway algorithm (31). We synthesized the 52 YF17D-derived peptides most likely to bind to Mamu-A*01 based on their predicted affinity for this MHC class I molecule. We then used a gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay to screen these peptides in YF17D-immunized animals at several time points after vaccination and discovered that four Mamu-A*01-binding peptides, LTPVTMAEV (LV9_1285-1293), VSPGNGWMI (VI9_3250-3258), MSPKGISRM (MM9_2179-2187), and TTPFGQQRVF (TF10_2853-2862), were recognized in vivo (Fig. ​(Fig.1C).1C). Using a previously reported protocol (26), we also observed CD8⁺ T-cell activation in all four animals (Fig. 1D and E). Thus, as was observed previously, the YF17D vaccine virus replicates in Indian rhesus monkeys (36) and induces neutralizing antibodies, yellow fever 17D-specific Mamu-A*01-restricted CD8⁺ T-cell responses, and CD8⁺ T-cell activation.Open in a separate windowFIG. 1.YF17D replicates and induces neutralizing antibodies, virus-specific CD8⁺ T cells, and the activation of CD8⁺ T cells in rhesus macaques. (A) Replication of YF17D during the first 10 days after vaccination with two different doses, as measured by quantitative PCR (Q-PCR) using the following primers: forward primer YF-17D 10188 (5′-GCGGATCACTGATTGGAATGAC-3′), reverse primer YF-17D 10264 (5′-CGTTCGGATACGATGGATGACTA-3′), and probe 6-carboxyfluorescein (6Fam)-5′-AATAGGGCCACCTGGGCCTCCC-3′-6-carboxytetramethylrhodamine (TamraQ). (B) Titer of neutralizing antibodies determined at 2 and 5 weeks after YF17D vaccination. (C) Fresh PBMC from vaccinees (100,000 cells/well) were used in IFN-γ ELISPOT assays (41) to assess T-cell responses against YF17D. We used 4 epitopes (LTPVTMAEV [LV9_1285-1293], VSPGNGWMI [VI9_3250-3258], MSPKGISRM [MM9_2179-2187], and TTPFGQQRVF [TF10_2853-2862]) predicted to bind to Mamu-A*01 as defined by the MHC pathway algorithm (31). All IFN-γ ELISPOT results were considered positive if they were ≥50 SFC/10⁶ PBMC and ≥2 standard deviations over the background. (D) Identification of activated CD8⁺ T cells after vaccination with YF17D based on the expression of the proliferation and proapoptotic markers Ki-67 and Bcl-2, respectively (26). We stained whole blood cells with antibodies against CD3 and CD8. We then permeabilized and subsequently labeled these cells with Bcl-2- and Ki-67-specific antibodies. The flow graphs were gated on CD3⁺ CD8⁺ lymphocytes. (E) Expression kinetics of Ki-67 and Bcl-2 in CD8⁺ T cells after vaccination with YF17D.We next engineered the YF17D vaccine virus to express amino acids 45 to 269 of SIVmac239 Gag (rYF17D/SIVGag_45-269) by inserting a yellow fever codon-optimized sequence between the genes encoding the viral proteins E and NS1. This recombinant virus replicated and induced neutralizing antibodies in mice (data not shown). We then tested the rYF17D/SIVGag_45-269 construct in six Mamu-A*01-positive Indian rhesus macaques. We found evidence for the viral replication of rYF17D/SIVGag_45-269 for five of these six macaques (Fig. ​(Fig.2A).2A). However, neutralizing antibodies were evident for all six animals at 2 weeks postvaccination (Fig. ​(Fig.2B).2B). Furthermore, all animals developed SIV-specific CD8⁺ T cells after a single immunization with rYF17D/SIVGag_45-269 (Fig. ​(Fig.2C).2C). To test whether a second dose of this vaccine could boost virus-specific T-cell responses, we administered rYF17D/SIVGag_45-269 (2.0 × 10⁵ PFU) to four macaques on day 28 after the first immunization and monitored cellular immune responses. With the exception of animal r04091, the rYF17D/SIVGag_45-269 boost did not increase the frequency of the vaccine-induced T-cell responses. This recombinant vaccine virus also induced CD8⁺ T-cell activation in the majority of the vaccinated animals (Fig. ​(Fig.2D2D).Open in a separate windowFIG. 2.rYF17D/SIVGag_45-269 replicates and induces neutralizing antibodies, virus-specific CD8⁺ T cells, and the activation of CD8⁺ T cells in rhesus macaques. (A) Replication of rYF17D/SIVGag_45-269 during the first 10 days after vaccination with two different doses as measured by Q-PCR using the YF17D-specific primers described in the legend of Fig. ​Fig.1.1. (B) Titer of neutralizing antibodies determined at 2 and 5 weeks after rYF17D/SIVGag_45-269 vaccination. The low levels of neutralization for animal r02013 were observed in three separate assays. (C) Fresh PBMC from vaccinees (100,000 cells/well) were used in IFN-γ ELISPOT assays to assess T-cell responses against the YF17D vector (red) and the SIV Gag(45-269) insert (black) at several time points postvaccination. We measured YF17D-specific responses using the same epitopes described in the legend of Fig. ​Fig.1.1. For SIV Gag-specific responses, we used 6 pools of 15-mers overlapping by 11 amino acids spanning the entire length of the SIVmac239 Gag insert. In addition, we measured Mamu-A*01-restricted responses against the dominant Gag_181-189CM9 and subdominant Gag_254-262QI9 epitopes. Four animals received a second dose of rYF17D/SIVGag_45-269 on day 28 after the first vaccination (dashed line). (D) Expression kinetics of Ki-67 and Bcl-2 in CD8⁺ T cells after vaccination with rYF17D/SIVGag_45-269. This assay was performed as described in the legend of Fig. ​Fig.11.We could not detect differences in vaccine-induced immune responses between the group of animals vaccinated with YF17D and the group vaccinated with rYF17D/SIVGag_45-269. There was, however, considerable animal-to-animal variability. Animal r02034, which was vaccinated with YF17D, exhibited massive CD8⁺ T-cell activation (a peak of 35% at day 14) (Fig. ​(Fig.1E),1E), which was probably induced by the high levels of viral replication (16,800 copies/ml at day 5) (Fig. ​(Fig.1A).1A). It was difficult to see differences between the neutralizing antibody responses induced by YF17D and those induced by rYF17D/SIVGag_45-269 (Fig. ​(Fig.1B1B and ​and2B).2B). However, neutralizing antibodies in animal r02013 decreased by 5 weeks postvaccination. It was also difficult to detect differences in the YF17D-specific CD8⁺ T-cell responses induced by these two vaccines. Peak Mamu-A*01-restricted CD8⁺ T-cell responses against YF17D ranged from barely detectable (animal r02110 at day 11) (Fig. ​(Fig.1C)1C) to 265 spot-forming cells (SFCs)/10⁶ peripheral blood mononuclear cells (PBMC) (animal r02034 at day 28) (Fig. ​(Fig.1C).1C). Similarly, three of the rYF17D/SIVGag_45-269-vaccinated animals (animals r04091, r04051, and r02013) made low-frequency CD8⁺ T-cell responses against the Mamu-A*01-bound YF17D peptides, whereas the other three animals (animals r03130, r02049, and r02042) recognized these epitopes with responses ranging from 50 to 200 SFCs/10⁶ PBMC (Fig. ​(Fig.2C).2C). For almost every rYF17D/SIVGag_45-269-vaccinated animal, the Gag_181-189CM9-specific responses (range, 50 to 750 SFCs/10⁶ PBMC) were higher than those generated against the Mamu-A*01-restricted YF17D epitopes (range, 0 to 175 SFCs/10⁶ PBMC), suggesting that the recombinant virus replicated stably in vivo (Fig. ​(Fig.2C).2C). Thus, the recombinant YF17D virus replicated and induced both virus-specific neutralizing antibodies and CD8⁺ T cells that were not demonstrably different from those induced by YF17D alone.Most viral vectors are usually more efficient after a prime with DNA or recombinant BCG (rBCG) (4, 11, 15, 18). We therefore used rYF17D/SIVGag_45-269 to boost two macaques that had been primed with rBCG expressing SIV proteins (Fig. ​(Fig.3A).3A). We detected no SIV-specific responses after either of the two priming rBCG vaccinations. Unfortunately, while the recombinant YF17D virus replicated well in animal r01056, we found evidence for only low levels of replication of rYF17D/SIVGag_45-269 on day 5 postvaccination for animal r01108 (7 copies/ml) (Fig. ​(Fig.3B).3B). Both animals, however, generated neutralizing antibodies at 2 weeks postvaccination (Fig. ​(Fig.3C).3C). Encouragingly, we detected high-frequency CD8⁺ T-cell responses in the Mamu-A*01-positive macaque (animal r01056) after boosting with rYF17D/SIVGag_45-269 (Fig. 3D to F). These responses were directed mainly against the Mamu-A*01-restricted Gag_181-189CM9 epitope, which is contained in the peptide pool Gag E (Fig. ​(Fig.3D).3D). Furthermore, the boost induced a massive activation of animal r01056''s CD8⁺ T cells, peaking at 35% at 17 days postvaccination (Fig. ​(Fig.3E).3E). Of these activated CD8⁺ T cells, approximately 10% were directed against the Gag_181-189CM9 epitope, with a frequency of 3.5% of CD8⁺ T cells (Fig. ​(Fig.3E).3E). These epitope-specific CD8⁺ T cells made IFN-γ, tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein 1β (MIP-1β), and degranulated (Fig. ​(Fig.3F3F and data not shown). Thus, an rBCG prime followed by a recombinant yellow fever 17D boost induced polyfunctional antigen-specific CD8⁺ T cells.Open in a separate windowFIG. 3.rYF17D/SIVGag_45-269 vaccination induced a robust expansion of Gag-specific responses in an rBCG-primed macaque. (A) Vaccination scheme. We immunized two rhesus macaques with rBCG intradermally (i.d.) (2.0 × 10⁵ CFU), rBCG orally (10⁷ CFU), and rYF17D/SIVGag_45-269 subcutaneously (2.0 × 10⁵ PFU) at 6-month intervals. rBCG was engineered to express 18 minigenes containing sequences of Gag, Vif, Nef, Rev, and Tat from SIVmac239. (B) Replication of rYF17D/SIVGag_45-269 during the first 10 days after vaccination as measured by Q-PCR using the YF17D-specific primers described in the legend of Fig. ​Fig.1.1. (C) Titer of neutralizing antibodies determined at 2 and 5 weeks after rYF17D/SIVGag_45-269 vaccination. (D) Fresh PBMC from animal r01056 (100,000 cells/well) were used in IFN-γ ELISPOT assays to assess T-cell responses against the YF17D vector (red) and the SIV Gag(45-269) insert (black) at several time points postvaccination. (E) Kinetics of CD8⁺ T-cell activation (as described in the legend of Fig. ​Fig.1)1) and expansion of Gag_181-189CM9-specific CD8⁺ T cells in animal r01056 after vaccination with rYF17D/SIVGag_45-269. (F) Vaccination with rYF17D/SIVGag_45-269 induced robust CD8⁺ T-cell responses against Gag_181-189CM9 in r01056. CD8⁺ T-cell activation (Ki-67⁺/Bcl-2⁻) for baseline and day 13 are shown. Gag_181-189CM9-specific responses were measured by tetramer staining and intracellular cytokine staining (ICS) with antibodies against MIP-1β and IFN-γ.Vaccine-induced CD8⁺ T cells are usually central memory T cells (T_CM) or effector memory T cells (T_EM). These two subsets of CD8⁺ T cells differ in function and surface markers (23). Repeated boosting drives CD8⁺ T cells toward the T_EM subset (23). We therefore determined whether a rBCG prime followed by a rYF17D/SIVGag_45-269 boost induced T_CM or T_EM CD8⁺ T cells. Staining of PBMC obtained on day 30 postvaccination revealed that the SIV-specific CD8⁺ T cells were largely T_EM cells since the majority of them were CD28 negative (Fig. ​(Fig.4A).4A). Furthermore, these cells persisted with the same phenotype until day 60 after vaccination (Fig. ​(Fig.4B).4B). It was recently suggested that T_EM cells residing in the mucosae can effectively control infection after a low-dose challenge with SIVmac239 (16).Open in a separate windowFIG. 4.rYF17D/SIVGag_45-269 vaccination of animal r01056 induced effector memory Gag_181-189CM9-specific CD8⁺ T cells that suppressed viral replication in CD4⁺ targets. (A and B) Frequency and memory phenotype of tetramer-positive Gag_181-189-specific CD8⁺ T cells in animal r01056 on day 30 (A) and day 60 (B) after rYF17D/SIVGag_45-269 vaccination. CD28 and CD95 expression profiles of tetramer-positive cells show a polarized effector memory phenotype. Cells were gated on CD3⁺ CD8⁺ lymphocytes. (C) Ex vivo Gag_181-189CM9-specific CD8⁺ T cells from animal r01056 inhibit viral replication from SIVmac239-infected CD4⁺ T cells. Gag_181-189CM9-specific CD8⁺ T cells from three SIV-infected Mamu-A*01-positive animals and rYF17D/SIVGag_45-269-vaccinated animal r01056 were tested for their ability to suppress viral replication from SIV-infected CD4⁺ T cells (39). Forty-eight hours after the incubation of various ratios of SIV-infected CD4⁺ T cells and Gag_181-189CM9-specific CD8⁺ T cells, the supernatant was removed and measured for viral RNA (vRNA) copies per ml by Q-PCR. We observed no suppression when effectors were incubated with CD4⁺ targets from Mamu-A*01-negative animals (data not shown). Animal rh2029 was infected with SIVmac239 (viral load, ∼10⁵ vRNA copies/ml) containing mutations in 8 Mamu-B*08-restricted epitopes as part of another study (37). Animal r01080 was vaccinated with a DNA/Ad5 regimen expressing Gag, Rev, Tat, and Nef and later infected with SIVmac239 (viral load, ∼10³ vRNA copies/ml) (42). Animal r95061 was vaccinated with a DNA/MVA regimen containing Gag_181-189CM9 and was later challenged with SIVmac239 (undetectable viral load) (2).We then assessed whether rYF17D/SIVGag_45-269-induced CD8⁺ T cells could recognize virally infected CD4⁺ T cells. We have shown that these vaccine-induced CD8⁺ T cells stain for tetramers and produce cytokines after stimulation with synthetic peptides (Fig. ​(Fig.3).3). None of these assays, however, tested whether these SIV-specific CD8⁺ T cells recognize SIV-infected cells and reduce viral replication. We therefore used a newly developed assay (39) to determine whether vaccine-induced CD8⁺ T cells can reduce viral replication in CD4⁺ T cells. We sorted tetramer-positive (Gag_181-189CM9-specific) lymphocytes directly from fresh PBMC and incubated them for 48 h with SIVmac239-infected CD4⁺ T cells expressing Mamu-A*01. We assessed the percentage of CD4⁺ T cells that expressed SIV Gag p27 (data not shown) and the quantity of virus in the culture supernatant (Fig. ​(Fig.4C).4C). Vaccine-induced CD8⁺ T cells reduced viral replication to the same extent as that seen with Gag_181-189CM9-specific CD8⁺ T cells purified from three SIVmac239-infected rhesus macaques, including an elite controller rhesus macaque, animal r95061 (Fig. ​(Fig.4C4C).The most encouraging aspect of this study is that rBCG primed a high-frequency CD8⁺ T-cell response after boosting with rYF17D/SIVGag_45-269. These CD8⁺ T cells reached frequencies that were similar to those induced by an rBCG prime followed by an Ad5 boost (11). Even without the benefit of the rBCG prime, the levels of CD8⁺ T cells induced by a single rYF17D/SIVGag_45-269 vaccination were equivalent to those induced by our best SIV vaccine, SIVmac239ΔNef. Recombinant YF17D generated an average of 195 SFCs/10⁶ PBMC (range, 100 to 750 SFCs/10⁶ PBMC) (n = 6), whereas SIVmac239ΔNef induced an average of 238 SFCs/10⁶ PBMC (range, 150 to 320 SFCs/10⁶ PBMC) (n = 3) (32). It is also possible that any YF17D/HIV recombinants would likely replicate better in humans than they have in rhesus macaques and thus induce more robust immune responses. Also, rBCG was shown previously to be effective in humans (5, 17, 33, 34) and may be more useful at priming T-cell responses in humans than it has been in our limited study with rhesus macaques. These two vectors have long-distinguished safety and efficacy histories in humans and may therefore be well suited for HIV vaccine development.