Genital CD8+ T cell response to HIV-1 gag in mice immunized by mucosal routes with a recombinant simian adenovirus

AdC6gag37, an E1-deleted adenovirus recombinant derived from the chimpanzee adenovirus serotype 6 expressing a codon-optimized truncated form of gag of HIV-1, was tested for induction of transgene-specific CD8+ T cell responses upon intranasal or intravaginal immunization of mice. Administration of AdC6gag37 induced gag-specific CD8+ T cells at systemic and mucosal sites. Frequencies of gag-specific CD8+ T cells elicited in the genital tract by intravaginal or intranasal immunizations were substantially increased by intranasal priming followed by intravaginal boosting with the same vector. Additionally, intravaginal immunization with AdC6gag37 increased the amount of gammadelta T cells that could be detected in genital tract.     

Enhanced CD8+ T cell response to HIV-1 env by combined immunization with influenza and vaccinia virus recombinants

With the aim to determine if immunization with two different live recombinant viral vectors could lead to an enhancement of the cellular immune response to HIV-1 antigens, we have characterized the CD8+ T cell response elicited against the V3 loop epitope from HIV-1 env protein in Balb/c mice immunized with either: a recombinant influenza virus (Flu-Env) expressing the V3 loop epitope from HIV-1 strain IIIB, a vaccinia virus recombinant (VV-Env) expressing the complete HIV-1-IIIB env protein, or a combination of both. The CD8+ T cell response, measured by the ELISPOT assay, in animals primed with Flu-Env and boosted with VV-Env was 5 to 6 times higher than in animals inoculated with either Flu-Env or VV-Env alone. Similar results were obtained with recombinant viruses expressing the V3 loop epitope or the complete env protein, respectively, from the MN strain of HIV-1. Our results indicate that the use of two different live vectors for priming and boosting has a synergistic effect on the immune response against HIV-1, and could represent a novel vaccination strategy against AIDS.     

Inhibitory receptor expression on memory CD8 T cells following Ad vector immunization

T cells are an important component of immune responses, and their function is influenced by their expression of inhibitory receptors. Immunization with alternative serotype adenovirus (Ad) vectors induces highly functional T cell responses with lower programmed cell death 1 (PD-1) expression and increased boostability relative to Ad5 vectors. However, a detailed phenotypic characterization of other inhibitory receptors is lacking, and it is unknown whether Ad5-induced CD8 T cells eventually recover function with time. In this report, we measure the expression of various inhibitory receptors and memory markers during early and late time points following vaccination with Ad5 and alternative serotype Ad vectors. CD8 T cells induced by Ad5 exhibited increased expression of the inhibitory receptor Tim-3 and showed decreased central memory differentiation as compared with alternative serotype Ad vectors, even a year following immunization. Moreover, relative to Ad5-primed mice, Ad26-primed mice exhibited substantially improved recall of SIV Gag-specific CD8 T cell responses following heterologous boosting with MVA or Ad35 vectors. We also demonstrate that low doses of Ad5 priming resulted in more boostable immune responses with lower PD-1 expression as compared to high Ad5 doses, suggesting a role for vector dose in influencing immune dysfunction following Ad5 vaccination. These data suggest that Ad5 vectors induce a long-term pattern of immune exhaustion that can be partly overcome by lowering vector dose and modulating inhibitory signals.     

Intramuscular rather than oral administration of replication-defective adenoviral vaccine vector induces specific CD8+ T cell responses in the gut

Gut-associated lymphoid tissue (GALT) is the primary replication site for HIV-1, resulting in a pronounced CD4(+) T cell loss in this tissue during primary infection. A mucosal vaccine that generates HIV-specific CD8(+) T cells in the gut could prevent the establishment of founder populations and broadcasting of virus. Here, we immunized mice orally and systemically with a chimpanzee derived adenoviral vector expressing HIV gag (AdC68gag) and measured frequencies of gag-specific interferon-gamma (IFN-gamma) producing CD8(+) T cells in the GALT. A single oral administration was inefficient at eliciting responses in the mesenteric lymph nodes and Peyer's Patches, while a single intramuscular administration elicited strong systemic and detectable mucosal responses. The gag-specific CD8(+) T cell responses were present in both acute and memory phases following intramuscular administration.     

Generation of antigen specific CD8+ cytotoxic T cells following immunization with soluble protein formulated with novel glycoside adjuvants.

Presentation of peptide on MHC class I molecules is essential to elicit cytolytic T cell (CTL) activity. Such peptides are a result of the cytosolic, or class I, antigen processing pathway. Due to the segregation of the class I and the exogenous processing pathway, soluble protein cannot enter the class I pathway and is thus incapable of inducing CTL. However careful formulation with adjuvants can overcome this obstacle. In this study we evaluated the capacity of two novel amphiphilic adjuvants, better termed delivery vehicles, to elicit CTL activity in a C57Bl/6 murine model with ovalbumin (OVA) as an antigen. Incomplete Freund's adjuvant and aluminium hydroxide (Alhydrogel) were used as reference adjuvants. In addition the oil-in-water emulsion Provax was used throughout as a positive control adjuvant. Both amphiphile preparations were capable of eliciting potent CTL activity after administration of one immunizing dose of ovalbumin. CTL were CD8+ restricted as assessed by in vitro depletion of CD8+ and CD4+ T cells. CTL activity was also MHC-restricted as well as specific for the H-2Kb OVA motif SIINFEKL.     

The functional consequences of delivery of HIV-1 Nef to dendritic cells using an adenoviral vector

The Nef gene is a major determinant of HIV-1 pathogenicity. Several immunomodulatory functions have been reported for Nef, including down-regulation of CD4 and class I MHC in T-lymphocytes, and the ability to enhance viral transmission from macrophages and dendritic cells (DC) to T-lymphocytes. In this study, HIV-1 (SF2 strain) Nef was expressed in human monocyte-derived dendritic cells, using an adenovirus based delivery system. Nef expression resulted in decreased CD4 levels, but no change to class I MHC, and no impairment in the ability of DC to stimulate recall PPD responses, mixed leukocyte responses, or hepatitis B-specific CD8 responses. The adenovirus vector itself stimulated a strong recall CD4 response in all individuals tested, and also induced up-regulation of class I MHC, CD86 and CD40 on the dendritic cell surface. The study provides no evidence that HIV Nef impairs the function of human dendritic cells, and suggests that delivery of Nef to dendritic cells may be one strategy with which to stimulate an HIV-1 immune response.     

Modified vaccinia Ankara expressing HIVA antigen stimulates HIV-1-specific CD8 T cells in ELISpot assays of HIV-1 exposed infants

Recombinant modified vaccinia virus Ankara expressing HIV-1 antigens (MVA.HIVA) was used in ELISpot assays to monitor HIV-1-specific T cell responses in infants. Responses to MVA.HIVA and HIV-1 peptides were examined in 13 infected and 81 exposed uninfected infants in Nairobi, Kenya. Responses to MVA.HIVA (38%) and peptide stimulation (38%) were similar in frequency (p=1.0) and magnitude (mean 176 versus 385 HIVSFU/10(6), p=0.96) in HIV-1 infected infants. In exposed uninfected infants, MVA.HIVA detected more positive responses and higher magnitude responses as compared to peptide. MVA.HIVA ELISpot is a sensitive method for quantification of HIV-1-specific CD8+ T cell responses in HIV-1 exposed infants. These results demonstrate the relevance of HIV-1 clade A consensus-derived immunogen HIVA for the viruses currently circulating in Nairobi.     

Immunization with an HIV-1 immunogen induces CD4+ and CD8+ HIV-1-specific polyfunctional responses in patients with chronic HIV-1 infection receiving antiretroviral therapy

Development of polyfunctional T lymphocyte responses is critical in the immunological response against HIV-1. Fifty-four HIV-1 infected patients receiving antiretroviral treatment (ART) and immunization with an HIV-1 immunogen or placebo, periodically every 3 months throughout a period of 36 months, were evaluated for the purposes of analysing the development of HIV-1-specific CD4⁺ and CD8⁺ responses. A significant increase of proliferating and IFN-γ producing CD8⁺ HIV-1-specific T cells, of HIV-1-specific precursor frequencies for CD8⁺ and for CD4⁺ T cells and of Gag/pol-specific memory CTL precursors (CTLp) was observed in the immunogen group in comparison to placebo. IL-2 intracellular expression and IFN-γ and TNF- co-expression in HIV-1-specific CD8⁺ T cells were also substantially increased in the immunized group. A negative correlation between viral load and CD3⁺CD4⁺CFSE^low HIV-1-specific lymphoproliferative response and frequency of Gag/pol-specific CTLp was solely observed in the HIV-1 immunogen group. Long-term immunization in patients receiving ART helps to develop HIV-1-specific polyfunctional T cell responses.     

Induction of specific CD8+ memory T cells and long lasting protection following immunization with Salmonella typhimurium expressing a lymphocytic choriomeningitis MHC class I-restricted epitope.

Numerous studies have shown the potential of Salmonella typhimurium as a vector for delivery of heterologous proteins for vaccination against other pathogens. Earlier studies showed that the inefficient elicitation of MHC class I-restricted responses could limit the use of S. typhimurium as a heterologous antigen delivery vector for vaccination. We recently developed an approach to overcome this limitation by using a bacterial-encoded specialized protein secretion system, termed type III, to deliver proteins into the class I antigen presenting pathways. Thus, peptides of interest fused to proteins bearing the type III secretion signal, which can elicit protective CTL responses. Because protective immunity is usually assessed a few weeks after vaccination, there is a paucity of information regarding duration of protective immunity induced by this system. We show here that mice immunized orally with S. typhimurium vectors expressing a MHC class I-restricted epitope of the lymphocytic choriomeningitis virus (LCMV) nucleoprotein developed specific antiviral CTL responses. CD8+ T cells were found to be necessary for this CTL activity against targets presenting the LCMV epitope. The survival of mice challenged with lethal doses of LCMV 60 or 135 days after vaccination was as complete as the survival of mice challenged 2 weeks after immunization with the same vectors. By demonstrating their ability to induce prolonged protective immunity after oral delivery, S. typhimurium vectors have met an essential requirement in support of their development as vectors for heterologous vaccination.     

Long-term increase of CD4+ central memory cells in HIV-1-infected individuals by therapeutic HIV-1 rgp160 immunization

OBJECTIVE: To evaluate functional potential and phenotypic markers in HIV-1-infected patients immunized with HIV-1 rgp160. METHODS: We assessed changes in T-cell phenotype and immune function in 12 HIV-1-infected individuals that were part of a therapeutic vaccine study from 1992 to 1995 [Sandstrom E, Wahren B. Therapeutic immunisation with recombinant gp160 in HIV-1 infection: a randomised double-blind placebo-controlled trial. Nordic VAC-04 Study Group. Lancet 1999;353(9166):1735-42]. The patients received 160mug HIV-1 rgp160 or placebo i.m. at baseline (day 0), and months 1, 2, 3, 4, 6, and thereafter every 3 months. Frozen peripheral blood mononuclear cells (PBMC) were retrieved from time points 0, 9, 12 and 24 months for phenotypic analysis utilizing flow cytometry. RESULTS: Up-regulation of immune activation markers HLA-DR and CD38 was observed at baseline and throughout the monitoring period on both CD4(+) and CD8(+) T cells in all patients, reflecting immune activation due to persistent high viral load. Further enhanced expression of activation markers was observed over time in the vaccine group, but not the placebo group. We also observed a consistent long-term increase of the CD4(+) central memory population (CD3(+)CD4(+)CD45RA(-)CCR7(+)) in the vaccinated group. CONCLUSIONS: Administration of eight doses of rgp160 in a year appeared to partially reverse some of the defects exerted by HIV-1 on the immune system. A combination of vaccination with effective antiretroviral therapy (ART) may thus represent an immunotherapeutic intervention for treatment of chronic HIV-1 infection. The improvement of a HIV-1-specific central memory population and HIV-1 antigen-specific CD4(+) lymphoproliferative responses may have contributed to the short-term improved survival reported in the vaccinated group.     

HIV感染者发生弥漫性CD8+淋巴细胞增多综合征的诊断

艾滋病毒(HIV)感染者的弥漫性浸润性淋巴细胞增多综合征(DILS)是以循环中CD8+T淋巴细胞增多为特征的.一些HIV感染者可能通过CD8+淋巴细胞的一种寡克隆扩增对感染作出反应.这些细胞浸润到多种器官,其中唾液腺和肺脏是主要累及部位.淋巴细胞浸润内脏器官的浸润过程与类sjogren-综合征类似,HIV感染者出现单纯腮腺肿大时,应该怀疑是否是DILS.此外,DILS肺部病变可能在临床及影像学表现与由卡氏肺囊虫引起的肺部病变相类似.DILS其它表现包括严重的周围神经病变、淋巴细胞浸润肝脏引起肝炎症状、肌炎和淋巴细胞性间质性肾炎等.     

Induction of cytotoxic T lymphocytes following immunization with cationized soluble antigen

Antigen presentation on major histocompatibility complex (MHC) class I and subsequent priming of antigen-specific cytotoxic T lymphocytes (CTLs) are essential steps for vaccination but exogenous soluble proteins are conventionally taken up by endosomes and presented on MHC class II rather than class I. In this study, we demonstrated, for the first time, that ovalbumin (OVA) chemically cationized with hexamethylenediamine (HMD) can induce OVA-specific CTLs without any adjuvants. Cationization of OVA greatly enhances cellular uptake by antigen-presenting cells (APCs) through adsorptive endocytosis. Two kinds of Cat-OVAs with different cationic charges were evaluated to elicit a CTL response through enhanced uptake by APCs and concomitant participation in the class I pathway. Cat(20)-OVA, a cationized OVA derivative with more cationic charges, showed pronounced induction of the OVA-specific CTL response after subcutaneous immunization. The CTL response was comparable with that induced by OVA with CFA. In contrast to the CFA formulation that actually produced local tissue damage in this study, local damage at the injection sites was not observed with Cat-OVAs. Cat(20)-OVA also showed a significant protective effect on the growth of OVA-expressing E.G7 tumor cells. In conclusion, cationization of soluble antigen is a useful and safe vaccination strategy.     

Booster immunization of antigen primed mice with anti-idiotypic T cells generates antigen-specific memory T cell response

Immunological memory is characterized by quick and enhanced immune response after re-exposure to the same antigen. In the present work, we have shown that anti-idiotypic T cells are generated in mice after immunization with idiotypic T cell clone or polyclonal T cell specific for nucleocapsid protein of Rinderpest virus. Further, we have shown that N specific idiotypic T cell receptors from apoptotic idiotypic T cells are processed by bone marrow derived dendritic cells and presented to cognate anti-idiotypic T cells. Evidence has been provided for the existence of antigen specific T cell idiotypic network in the body. Boosting with antigen specific anti-idiotypic T cells generates memory response in the antigen-primed mice.     

Mutant Escherichia coli enterotoxin as a mucosal adjuvant induces specific Th1 responses of CD4+ and CD8+ T cells to nasal killed-bacillus calmette-guerin in mice

On single nasal immunization of mice with killed-bacillus calmette-guerin (BCG) plus a mutant Escherichia coli enterotoxin, delayed-type hypersensitivity was induced and BCG-infection decreased. Spleen cells, particularly CD4+ T cells among them produced IL-2, IFNgamma and TNFalpha in response to the killed-BCG or purified protein derivatives. CD8+ T cells including cytotoxic T lymphocytes produced IFNgamma and TNFalpha. However, both types of T cells reacted a little to Ag85B. The mutant induces cellular immunity to nasal killed-BCG vaccine and decreases BCG-infection. CD4+ and CD8+ T cells produce cytokines effective for tuberculosis. Although killed-BCG loses some antigens like Ag85B, nasal killed-BCG plus the mutant is useful for tuberculosis.     

Control of HIV-1 replication in vitro by vaccine-induced human CD8+ T cells through conserved subdominant Pol epitopes

ObjectiveThe specificity of CD8⁺ T cells is critical for early control of founder/transmitted and reactivated HIV-1. To tackle HIV-1 variability and escape, we designed vaccine immunogen HIVconsv assembled from 14 highly conserved regions of mainly Gag and Pol proteins. When administered to HIV-1-negative human volunteers in trial HIV-CORE 002, HIVconsv vaccines elicited CD8⁺ effector T cells which inhibited replication of up to 8 HIV-1 isolates in autologous CD4⁺ cells. This inhibition correlated with interferon-γ production in response to Gag and Pol peptide pools, but direct evidence of the inhibitory specificity was missing. Here, we aimed to define through recognition of which epitopes these effectors inhibit HIV-1 replication.DesignCD8⁺ T-cells from the 3 broadest HIV-1 inhibitors out of 23 vaccine recipients were expanded in culture by Gag or Pol peptide restimulation and tested in viral inhibition assay (VIA) using HIV-1 clade B and A isolates.MethodsFrozen PBMCs were expanded first using peptide pools from Gag or Pol conserved regions and tested on HIV-1-infected cells in VIA or by individual peptides for their effector functions. Single peptide specificities responsible for inhibition of HIV-1 replication were then confirmed by single-peptide expanded effectors tested on HIV-1-infected cells.ResultsWe formally demonstrated that the vaccine-elicited inhibitory human CD8⁺ T cells recognized conserved epitopes of both Pol and Gag proteins. We defined 7 minimum epitopes, of which 3 were novel, presumably naturally subdominant. The effectors were oligofunctional producing several cytokines and chemokines and killing peptide-pulsed target cells.ConclusionsThese results implicate the use of functionally conserved regions of Pol in addition to the widely used Gag for T-cell vaccine design. Proportion of volunteers developing these effectors and their frequency in circulating PBMC are separate issues, which can be addressed, if needed, by more efficient vector and regimen delivery of conserved immunogens.     

HIV-1感染者CD8+T细胞受体基因多样性特点及其与病毒载量的相关性

目的 研究HIV-1感染者CD8+T细胞受体(TCR)基因的多样性变化特征及其与病毒载量的相关性.方法 应用抗CD8单克隆抗体从9份HIV-1感染者和7份HIV-1阴性对照样本外周血单个核细胞中分离CD8+T细胞,提取总RNA,然后采用一步(one step)及巢式(nested)多聚酶链式反应(PCR)的方法对22个T细胞受体Vβ基因家族的瓦补决定区3(CDR3)进行扩增,利用ABI-3700测序仪对扩增的PCR产物进行扣描,定量分析HIV-1感染者TCR CDR3区多样性变化特征及其与病毒载量的相关性.结果 HIV-1感染者和正常人相比较其CD8+T细胞的TCR基因多样性明显减少,部分TCR Vβ基因家族CDR3区的高斯分布破坏;TCR的紊乱与病毒载量呈正相关(r=0.771,P<0.05);HIV-1感染引起患者TCR多样性的改变不仅表现在不同Vβ基因家族上,而且也表现在CDR3长度上,其中感染者VB2、Vβ4、Vβ5、Vβ17、Vβ20、Vβ21、Vβ23及Vβ24基因家族的变化与正常人存在统计学差异.结论 HIV-1感染能引起CD8+T细胞TCR基因多样性的减少及高斯分布的破坏,TCR CDR3区的紊乱与病毒载量呈正相关.     

Intranasal immunization with vaccine vector Streptococcus gordonii elicits primed CD4 and CD8 T cells in the genital and intestinal tracts

Generation of primed T cells is crucial for the development of optimal vaccination strategies. Using a TCR-transgenic CD4⁺ and CD8⁺ T cell adoptive transfer model, we demonstrate that a single nasal immunization with recombinant Streptococcus gordonii induces antigen-specific primed T cells in lymph nodes draining the genital and intestinal tracts with about 80% of CD4⁺ and 50% of CD8⁺ proliferating cells. T cell clonal expansion was also observed in cervical lymph nodes, draining the immunization site, and in the spleen. The modulation of CD44 and CD45RB marker expression indicated that proliferating T cells were activated. Proliferation in distal mesenteric and iliac lymph nodes and in the spleen was observed 5 days after nasal immunization, while in draining cervical lymph nodes proliferation peaked already at day 3. The division profile of transgenic T cells observed in iliac and mesenteric lymph nodes was discontinuous, showing the lack of early cell divisions. The kinetics of T cell clonal expansion, the discontinuous division profile and the modulation of migration markers such as CD62L suggest that activated antigen-specific T cells disseminate from the immunization site to distal intestinal and genital tracts.     

Estradiol-induced vaginal mucus inhibits antigen penetration and CD8 T cell priming in response to intravaginal immunization

Although vaginal immunization has been explored as a strategy to induce mucosal immunity in the female reproductive tract, this site displays unique immunological features that probably evolved to inhibit anti-paternal T cell responses after insemination to allow successful pregnancy. We previously demonstrated that estradiol, which induces an estrus-like state, prevented CD8⁺ T cell priming during intravaginal immunization of mice. We now show that estradiol prevented antigen loading of vaginal antigen presenting cells (APCs) after intravaginal immunization. Histological examination confirmed that estradiol prevented penetration of peptide antigen into the vaginal wall. Removal of the estradiol-induced mucus barrier by mucinase partially restored antigen loading of vaginal APC and CD8⁺ T cell proliferation in vivo. The estradiol-induced mucus barrier may thus prevent exposure to antigens delivered intravaginally, supplementing additional estradiol-dependent mechanism(s) that inhibit CD8⁺ T cell priming after insemination or vaginal vaccination.     

CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

Human immunodeficiency virus type 1 (HIV-1) canarypox vaccines are safe but poorly immunogenic. CD40 ligand (CD40L), a member of the tumor necrosis factor superfamily (TNFSF), is a pivotal costimulatory molecule for immune responses. To explore whether CD40L can be used as an adjuvant for HIV-1 canarypox vaccine, we constructed recombinant canarypox viruses expressing CD40L. Co-immunization of mice with CD40L expressing canarypox and the canarypox vaccine expressing HIV-1 proteins, vCP1452, augmented HIV-1 specific cytotoxic T lymphocyte (CTL) responses in terms of frequency, polyfunctionality and interleukin (IL)-7 receptor alpha chain (IL-7Ralpha, CD127) expression. In addition, CD40L expressed from canarypox virus could significantly augment CD4+ T cell responses against HIV-1 in mice. CD40L expressed from canarypox virus matured human monocyte-derived dendritic cells (MDDCs) in a tumor necrosis factor-alpha (TNF-alpha) independent manner, which underwent less apoptosis, and could expand ex vivo Epstein-Barr virus (EBV)-specific CTL responses from healthy human individuals and ex vivo HIV-1-specific CTL responses from HIV-1-infected individuals in the presence or absence of CD4+ T cells. Taken together, our results suggest that CD40L incorporation into poxvirus vectors could be used as a strategy to enhance their immunogenicity.     

CD4~+/CD8~+T细胞比值在HIV-1母婴传播早期诊断的应用

目的应用CD4+/CD8+T细胞(CD4/CD8)比值对母婴传播婴幼儿人类免疫缺陷病毒(HIV)感染进行早期诊断,为临床应用提供依据。方法对2011-2013年HIV阳性孕妇的115例3~11个月龄婴幼儿收集抗凝全血进行CD4+T细胞和HIV-1DNA检测。结果 115例婴幼儿中,检出HIV-1DNA 5例,检出率4.34%,且CD4+/CD8+T细胞比值均1.0。115例中CD4+%均25%。在110例HIV-1DNA阴性中有2例CD4+T细胞绝对数1 500细胞/μl。5例HIV-1DNA阳性中1例CD4+T细胞绝对数1 500细胞/μl,4例CD4+T细胞绝对数1 500细胞/μl。结论婴幼儿的CD4/CD8比值1.0是诊断发生母婴传播的有效早期诊断方法。