Induction and maintenance of a phenotypically heterogeneous lung tissue-resident CD4+ T cell population following BCG immunisation

Tuberculosis (TB) is the biggest cause of human mortality from an infectious disease. The only vaccine currently available, bacille Calmette-Guérin (BCG), demonstrates some protection against disseminated disease in childhood but very variable efficacy against pulmonary disease in adults. A greater understanding of protective host immune responses is required in order to aid the development of improved vaccines. Tissue-resident memory T cells (T_RM) are a recently-identified subset of T cells which may represent an important component of protective immunity to TB. Here, we demonstrate that intradermal BCG vaccination induces a population of antigen-specific CD4⁺ T cells within the lung parenchyma which persist for >12 months post-vaccination. Comprehensive flow cytometric analysis reveals this population is phenotypically and functionally heterogeneous, and shares characteristics with lung vascular and splenic CD4⁺ T cells. This underlines the importance of utilising the intravascular staining technique for definitive identification of tissue-resident T cells, and also suggests that these anatomically distinct cellular subsets are not necessarily permanently resident within a particular tissue compartment but can migrate between compartments. This lung parenchymal population merits further investigation as a critical component of a protective immune response against Mycobacterium tuberculosis (M. tb).     

The association between parental and neonatal BCG vaccination and neonatal T helper 17 cell expansion

BackgroundBacille Calmette-Guérin (BCG) vaccination reduces the severity of neonatal infections; this effect appears enhanced if the mother has received BCG. We performed immunophenotyping of the T-cell subset and characterized T-cell proliferation responses to assess possible immune response pathways.MethodsHealthy BCG-vaccinated (n = 8) and unvaccinated (n = 9) neonates born by elective caesarean section were sampled 3 weeks after birth. We compared a wide panel of intracellular cytokine and cell surface expression markers as well as proliferation response in T-cells between BCG-vaccinated and unvaccinated neonates, stratified by parental BCG status.ResultsFor all BCG-vaccinated neonates and 3 of 9 unvaccinated neonates that served as controls, both parents had a BCG scar. Th17 (CD4 + IL-17+) prevalence as percentage of total CD4 + T-cells was expanded 4-fold in BCG-vaccinated compared to unvaccinated, being 11.6% [3.6–19.6%] vs 2.8% [1.0–6.6%]. Th17 counts for 3 unvaccinated neonates born to BCG-vaccinated parents was comparable to vaccinated neonates, and higher than remaining controls, parental BCG = 8.5% [4.4–8.9%] vs 1.8% [0.8–3.3%] for no parental BCG (median [interquartile range] for all data).ConclusionAmong neonates born to BCG-vaccinated parents, the prevalence of Th17 cells, important in the response against bacterial infections, was substantially elevated. The interaction between neonatal and parental BCG for Th17 responses and the importance remains to be further investigated.     

The novel tuberculosis vaccine,AERAS-402, is safe in healthy infants previously vaccinated with BCG,and induces dose-dependent CD4 and CD8T cell responses

Background

Efforts to reduce risk of tuberculosis disease in children include development of effective vaccines. Our aim was to test safety and immunogenicity of the new adenovirus 35-vectored tuberculosis vaccine candidate AERAS-402 in infants, administered as a boost following a prime with the Bacille Calmette-Guerin vaccine.

Methods

In a phase 1 randomised, double-blind, placebo-controlled, dose-escalation trial, BCG-vaccinated infants aged 6–9 months were sequentially assigned to four study groups, then randomized to receive an increasing dose-strength of AERAS-402, or placebo. The highest dose group received a second dose of vaccine or placebo 56 days after the first. The primary study outcome was safety. Whole blood intracellular cytokine staining assessed immunogenicity.

Results

Forty-two infants received AERAS-402 and 15 infants received placebo. During follow-up of 182 days, an acceptable safety profile was shown with no serious adverse events or discontinuations related to the vaccine. AERAS-402 induced a specific T cell response. A single dose of AERAS-402 induced CD4T cells predominantly expressing single IFN-γ whereas two doses induced CD4T cells predominantly expressing IFN-γ, TNF-α and IL-2 together. CD8T cells were induced and were more likely to be present after 2 doses of AERAS-402.

Conclusions

AERAS-402 was safe and immunogenic in healthy infants previously vaccinated with BCG at birth. Administration of the highest dose twice may be the most optimal vaccination strategy, based on the induced immunity. Multiple differences in T cell responses when infants are compared with adults vaccinated with AERAS-402, in the same setting and using the same whole blood intracellular cytokine assay, suggest specific strategies may be important for vaccination for each population.     

Enhancement of antigen-specific CD4+ and CD8+ T cell responses using a self-assembled biologic nanolipoprotein particle vaccine

To address the need for vaccine platforms that induce robust cell-mediated immunity, we investigated the potential of utilizing self-assembling biologic nanolipoprotein particles (NLPs) as an antigen and adjuvant delivery system to induce antigen-specific murine T cell responses. We utilized OT-I and OT-II TCR-transgenic mice to investigate the effects of NLP-mediated delivery of the model antigen ovalbumin (OVA) on T cell activation. Delivery of OVA with the TLR4 agonist monophosphoryl lipid A (MPLA) in the context of NLPs significantly enhanced the activation of both CD4⁺ and CD8⁺ T cells in vitro compared to co-administration of free OVA and MPLA. Upon intranasal immunization of mice harboring TCR-transgenic cells, NLPs enhanced the adjuvant effects of MPLA and the in vivo delivery of OVA, leading to significantly increased expansion of CD4⁺ and CD8⁺ T cells in lung-draining lymph nodes. Therefore, NLPs are a promising vaccine platform for inducing T cell responses following intranasal administration.     

Assessment of SARS-CoV-2 specific CD4(+) and CD8 (+) T cell responses using MHC class I and II tetramers

The success of SARS-CoV-2 (CoV-2) vaccines is measured by their ability to mount immune memory responses that are long-lasting. To achieve this goal, it is important to identify surrogates of immune protection, namely, CoV-2 MHC Class I and II immunodominant pieces/epitopes and methodologies to measure them. Here, we present results of flow cytometry-based MHC Class I and II QuickSwitch^TM platforms for assessing SARS-CoV-2 peptide binding affinities to various human alleles as well as the H-2 Kb mouse allele. Multiple SARS-CoV-2 potential MHC binders were screened and validated by QuickSwitch testing. The screen included 31 MHC Class I and 19 MHC Class II peptides predicted to be good binders by the IEDB web resource provided by NIAID. While several predicted peptides with acceptable theoretical Kd showed poor MHC occupancies, fourteen MHC class II and three MHC class I peptides showed promiscuity in that they bind to multiple MHC molecule types. In addition to providing important data towards the study of the SARS-CoV-2 virus and its presented antigenic epitopes, the peptides identified in this study can be used in the QuickSwitch platform to generate MHC tetramers. With those tetramers, scientists can assess CD4 + and CD8 + immune responses to these different MHC/peptide complexes.     

Vaccine-induced anti-tuberculosis protective immunity in mice correlates with the magnitude and quality of multifunctional CD4 T cells

The development of improved vaccines against Mycobacterium tuberculosis has been hindered by a limited understanding of the immune correlates of anti-tuberculosis protective immunity. In this study, we examined the relationship between long-term anti-tuberculosis protection and the mycobacterial-specific CD4 multifunctional T (MFT) cell responses induced by five different TB vaccines (live-attenuated, subunit, viral vectored, plasmid DNA, and combination vaccines) in a mouse model of pulmonary tuberculosis. In a 14-month experiment, we showed that TB vaccine-induced CD4 T cell responses were heterogenous. Antigen-specific monofunctional CD4 T cells expressing single cytokines and MFT CD4 T cells expressing multiple cytokines (IFN-γ and TNF-α, IFN-γ and IFN-γ, TNF-α, and IL-2, and all three cytokines) were identified after the immunizations. Interestingly, compared to the monofunctional cells, significantly higher median fluorescent intensities (MFIs) for IFN-γ and TNF-α were detected for triple-positive MFT CD4 T cells induced by the most protective vaccines while modest differences in relative MFI values were seen for the less protective preparations. Most importantly during the 14-month study, the levels of vaccine-induced pulmonary and splenic protective immunity correlated with the frequency and the integrated MFI (iMFI, frequency × MFI) values of triple-positive CD4 T cells that were induced by the same vaccines. These data support efforts to use MFT cell analyses as a measure of TB vaccine immunogenicity in human immunization studies.     

Associations between helminth infection and CD4+ T cell count,viral load and cytokine responses in HIV-1-infected Ugandan adults

It has been proposed that helminth infection may exacerbate HIV progression by promoting activation of 'type 2' immune responses. To examine this hypothesis, we investigated helminth infection in a cohort of HIV-1-seropositive adults in Entebbe, Uganda, during November 1999 to January 2000. Individuals with helminths were treated. At enroLlment, after 5 weeks and after 4 months, CD4+ and CD8+ T cell counts and viral load were measured. Cytokine responses (interferon [IFN]-gamma, interleukin [IL]-2, IL-4 and IL-5) to Schistosoma mansoni adult worm antigen (SWA), Mycobacterium tuberculosis culture filtrate proteins (CFPs) and phytohaemagglutinin (PHA) were measured in a whole blood assay. At baseline, CD4+ T cell counts and CD4+: CD8+ ratios were higher in individuals with helminths than in those without (median CD4+ T cell counts 467/microL and 268/microL, respectively, P = 0.005). Viral load was lower among those with helminths but this was not statistically significant. During follow-up, CD4+ T cell counts and cytokine responses to PHA fell among individuals without helminths. Among those treated for helminths, CD4+ counts remained stable. Viral loads showed a transient increase at 5 weeks, which was more marked among those treated for helminths, but the levels at 4 months were similar to baseline in both groups. Among those with schistosomiasis, IFN-gamma and IL-2 responses to CFP, and IL-2 and IL-4 responses to PHA declined but there was a sustained increase in cytokine responses to SWA following treatment. These data do not support the hypothesis that helminth infection exacerbates HIV infection. The possibility that chronic helminth infection may suppress HIV replication and that effects on HIV replication may vary during helminth infection and treatment should be considered.     

不同免疫状态下启动HARRT后的CD4~+T淋巴细胞重建效果分析

目的了解浦东新区医学随访管理艾滋病毒感染者/艾滋病患者(HIV/AIDS)的早治疗情况,分析在不同CD4~+T淋巴细胞计数水平下启动高效抗逆转录病毒治疗(HAART)后的免疫重建效果。方法采用历史队列研究的方法,收集2004年1月至2016年7月期间在上海市浦东新区启动HAART的HIV/AIDS人群的人口学特征、CD4~+T淋巴细胞检测时间、检测结果等信息进行统计描述与分析。结果共入组953名对象,研究对象启动HARRT时的CD4~+T淋巴细胞计数水平呈逐年升高的趋势,早治疗的比例逐年升高。CD4~+T淋好巴细胞计数随着治疗时间的延长而增加,基线CD4~+T淋巴细胞计数在500个/μL以下对象的CD4~+T淋巴细胞计数与治疗时间呈明显的线性关系,对象之间的CD4上升幅度比较接近;基线CD4~+T淋巴细胞计数500个/μL对象的CD4~+T淋巴细胞计数与治疗时间呈不明显的线性关系,治疗12个月后CD4的上升幅度有所下降。基线CD4~+T淋巴细胞计数水平较高的对象启动HAART后CD4~+T淋巴细胞计数也始终维持在较高水平。结论晚治疗HIV/AIDS的免疫功能虽然可以不断恢复,但免疫状态始终处于相对较低的水平,通过早治疗可以一直保持较高的免疫水平,因此应进一步提高早治疗的比例。     

Ovalbumin lipid core peptide vaccines and their CD4 and CD8 T cell responses

The lipid core peptide (LCP) system has successfully been used in development of peptide-based vaccines against cancer and infectious diseases (such as group A streptococcal infection). CD8⁺ T cells are important targets for vaccines, however developing a vaccine that activates long-lasting immunity has proven challenging. The ability of LCP vaccines to activate antigen-specific CD8⁺ and/or CD4⁺ T cell responses was tested using compounds that contained two or four copies of OVA_257–264 and/or OVA_323–339 peptides conjugated to LCP, which are recognised by OTI (CD8⁺ specific) and OTII (CD4⁺ specific) T cells, respectively. The LCP–ovalbumin vaccines developed in this study were synthesised in 30% yields and showed no significant haemolytic effect on red blood cells (below 4% haemolysis when tested with compounds at up to 100 μM concentrations). Promising in vivo data in mice suggested that this LCP–ovalbumin vaccine system could act as a novel and potent vehicle for the stimulation of robust antigen-specific CD8⁺ T cell responses.     

The improved antibody response against HIV-1 after a vaccination based on intrastructural help is complemented by functional CD8+ T cell responses

Despite more than three decades of intense research, a prophylactic HIV-1 vaccine remains elusive. Four vaccine modalities have been evaluated in clinical efficacy studies, but only one demonstrated at least modest efficacy, which correlated with polyfunctional antibody responses to the HIV surface protein Env. To be most effective, a HIV-1 vaccine probably has to induce both, functional antibody and CD8⁺ T cell responses. We therefore analyzed DNA/DNA and DNA/virus-like particle (VLP) regimens for their ability to induce humoral and cellular immune responses. Here, DNA vaccination of mice induced strong CD8⁺ responses against Env and Gag. However, the humoral response to Env was dominated by IgG1, a subclass known for its low functionality. In contrast, priming only with the Gag-encoding plasmid followed by a boost with VLPs consisting of Gag and Env improved the quality of the anti-Env antibody response via intrastructural help (ISH) provided by Gag-specific T cells to Env-specific B cells. Furthermore, the Gag-specific CD8⁺ T cells induced by the DNA prime immunization could still protect from a lethal infection with recombinant vaccinia virus encoding HIV Gag. Therefore, this immunization regimen represents a promising approach to combine functional antibody responses toward HIV Env with strong CD8⁺ responses controlling early viral replication.     

A combined carrier-adjuvant system of peptide nanofibers and toll-like receptor agonists potentiates robust CD8+ T cell responses

Improving CD8+ T cell responses activated by subunit vaccination is crucial for improving vaccine efficacy and safety. Here we report a carrier-adjuvant system composed of self-assembling peptide nanofibers presenting an immunodominant antigen from herpes simplex virus (HSV) and toll-like receptor (TLR) agonists that induces robust effector and memory CD8+ T cell responses in mice. The effector function of vaccine-induced CD8+ T cells was influenced by the type of TLR agonist. The use of CpG (TLR9 agonist) resulted in significantly greater specific in vivo cytotoxicity and trended towards more cells producing both IFN-γ and TNF-α compared to gardiquimod (TLR7 agonist). Prime-boost immunization with peptide nanofibers combined with either adjuvant resulted in development of HSV-specific CD8+ memory T cells further demonstrating the capability of the carrier-adjuvant system to induce strong HSV-specific CD8+ T cell responses. Inclusion of peptide epitope-nanofibers in protein-based subunit vaccines should increase the functional spectrum of the vaccine-elicited immune response and protection.     

HAART治疗AIDS患者基线CD4^+细胞水平对免疫重建效果的预测价值

目的探讨高效抗逆转录病毒治疗(HAART)AIDS患者基线CD4+细胞计数和CD4/CD8水平对免疫重建效果的预测价值。方法选取2008年6月至2017年12月于南京市第二人民医院完成24个月HAART治疗且病毒载量<50拷贝/毫升的患者共90例纳入研究,根据其治疗完成后CD4+细胞数分为3组:免疫无反应者(INRs)(<200个/μL),免疫应答不足(IIRs)(200~500个/μL)和免疫应答(IRs)(>500个/μL);比较3组之间的特征及动态CD4+细胞计数,进行免疫重建的因素分析。结果3组基本特征比较发现,CD4+细胞基线值与确认HIV至HAART治疗开始时间的方面差异具有统计学意义(P<0.05)。经抗病毒治疗后,IRs组CD4+细胞计数明显高于INRs和IIRs组,差异具有统计学意义(P<0.001)。ROC曲线下面积表明HAART治疗1年时CD4+细胞数达到最高预测价值。结论早期开启HAART可以减少免疫重建失败率。结合CD4+细胞基线数和CD4/CD8比值可以作为一个有效预测HAART治疗后免疫重建诊断的指标。     

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。     

Delayed BCG vaccination results in minimal alterations in T cell immunogenicity of acellular pertussis and tetanus immunizations in HIV-exposed infants

BackgroundBacille Calmette–Guerin (BCG) is effective in preventing disseminated tuberculosis (TB) in children but may also have non-specific benefits, and is thought to improve immunity to unrelated antigens through trained innate immunity. In HIV-infected infants, there is a risk of BCG-associated adverse events. We aimed to explore whether delaying BCG vaccination by 8 weeks, in utero or perinatal HIV infection is excluded, affected T-cell responses to B. pertussis (BP) and tetanus toxoid (TT), in HIV-exposed, uninfected infants.MethodsInfants were randomized to receive BCG vaccination at birth or 8 weeks of age. At 8 and 14 weeks, T cell proliferation and intracellular cytokine (IL-2, IL-13, IL-17, and IFN-γ) expression was analyzed in response to BP, TT and Staphylococcal enterotoxin B (SEB) antigens.ResultsDelaying BCG vaccination did not alter T-cell proliferation to BP or TT antigens. Infants immunized with BCG at birth had higher CD4+ T cell proliferation to SEB at 14 weeks of age (p = 0.018). Birth-vaccinated infants had increased CD8+ IL-2 expression in response to BP, but not TT or SEB, at 8 weeks. Infants vaccinated with BCG at 8 weeks had significantly lower IL-13 expression by BP-specific CD4+ and CD8+ T cells at 14 weeks (p = 0.032 and p = 0.0035, respectively). There were no observed differences in multifunctional cytokine response to TT, BP or SEB between infants vaccinated with BCG at birth versus 8 weeks of age.ConclusionDelaying BCG vaccination until 8 weeks of age results in robust T-cellular responses to BP and TT in HIV-exposed infants.Clinical Trial Registry: NCT02062580.     

Induction of mucosal HIV-specific B and T cell responses after oral immunization with live coxsackievirus B4 recombinants

Given the limited success of clinical HIV vaccine trials, new vaccine strategies are needed for the HIV pipeline. The present study explored the novel concept that a live enteric virus, with limited disease potential, is a suitable vaccine vector to elicit HIV-specific immune responses in the gut mucosa of immunized mice. Two coxsackievirus B4 (CVB4) vaccine vectors were designed to induce HIV-specific B or T cell responses. A B cell immunogen, CVB4/gp41(2F5), was constructed by expressing an epitope from the membrane proximal external region (MPER) of gp41 as a structural peptide within a surface loop of a capsid protein of CVB4. The T cell immunogen, CVB4/p24(73(3)), was constructed previously by expressing a gag p24 sequence as a non-structural peptide at the amino-terminus of the CVB4 polyprotein. The CVB4/gp41(2F5) recombinant was antigenic in mice and elicited anti-gp41 antibodies in both the mucosal and systemic compartments. The route of immunization affected the antibody response since oral delivery of CVB4/gp41(2F5) induced anti-gp41 antibodies in the mucosal but not in the systemic compartment while parenteral delivery induced anti-gp41 antibodies in both compartments. In contrast, oral immunization with CVB4/p24(73(3)) elicited both mucosal and systemic gag p24-specific T cell responses. Since coxsackieviruses are ubiquitous in the human population, a key question is whether pre-existing vector immunity will inhibit the ability of a CVB4-based vaccine to induce HIV-specific immune responses. We show that pre-existing vector immunity did not preclude the development of mucosal anti-gp41 antibodies or gag p24-specific T cell responses after oral immunization with the CVB4/HIV recombinants. We suggest that the CVB4/HIV recombinants have the potential to be a viable vaccine product because of ease of delivery, safety, immunogenicity, ease of large-scale production, and storage conditions requiring cold-chain temperatures provided by refrigeration.     

A novel HSV-2 subunit vaccine induces GLA-dependent CD4 and CD8 T cell responses and protective immunity in mice and guinea pigs

Background/objectivesThere is currently no licensed prophylactic or therapeutic vaccine for HSV-2 infection.MethodsWe developed a novel preclinical vaccine candidate, G103, consisting of three recombinantly expressed HSV-2 proteins (gD and the UL19 and UL25 gene products) adjuvanted with the potent synthetic TLR4 agonist glucopyranosyl lipid A (GLA) formulated in stable emulsion. The vaccine was tested for immunogenicity and efficacy in pre-clinical models for preventative and therapeutic vaccination.ResultsVaccination of mice with G103 elicited antigen-specific binding and neutralizing antibody responses, as well as robust CD4 and CD8 effector and memory T cells. The T cell responses were further boosted by subsequent challenge with live virus. Prophylactic immunization completely protected against lethal intravaginal HSV-2 infection in mice, with only transient replication of virus in the genital mucosa and sterilizing immunity in dorsal root ganglia. Supporting the use of G103 therapeutically, the vaccine expanded both CD4 and CD8 T cells induced in mice by previous infection with HSV-2. In the guinea pig model of recurrent HSV-2 infection, therapeutic immunization with G103 was approximately 50% effective in reducing the number of lesions per animal as well as the overall lesions score.ConclusionsTaken together, the data show that G103 is a viable candidate for development of a novel prophylactic and therapeutic HSV-2 vaccine.     

Co-infection with subclinical HIV and Wuchereria bancrofti, and the role of malaria and hookworms, in adult Tanzanians: infection intensities, CD4/CD8 counts and cytokine responses

The association between HIV and Wuchereria bancrofti, and the role of malaria and hookworms, were analysed by comparing three groups of individuals with: (1) HIV (HIV+; n=16); (2) W. bancrofti (circulating filarial antigen (CFA)+; n=25); and (3) HIV and W. bancrofti (HIV+/CFA+; n=18). A slightly higher HIV load and lower CD4% was observed in the HIV+/CFA+ group compared with the HIV+ group, and a slightly higher W. bancrofti CFA intensity was observed in the CFA+ group compared with the HIV+/CFA+ group, but none of these differences were statistically significant. Specific and non-specific IL-4, IL-10, IFNgamma and TNF levels were measured. Only specific IL-4 was significantly higher in the CFA+ group compared with the HIV+/CFA+ group. Thus, there was no clear evidence for an interaction between HIV and W. bancrofti infection. A multiple linear regression model showed that the presence of CFA was strongly positively associated with specific TNF response and, similarly, that HIV-positive individuals had higher TNF responses than HIV-negative individuals. Interestingly, the CD4% and CD4/CD8 ratio were higher in HIV-positive individuals with hookworms than in those without hookworm co-infection. Malaria was not associated with any of the other infections, or with CD4/CD8 counts or cytokine responses.     

Generation of a universal CD4 memory T cell recall peptide effective in humans,mice and non-human primates

CD4T cells play a key role in humoral immunity by providing help to B cells, enabling effective antibody class switching and affinity maturation. Some vaccines may generate a poor response due to a lack of effective MHC class II epitopes, resulting in ineffective helper T cell activation and recall and consequently poor humoral immunity. It may be beneficial to provide a CD4 T cell helper peptide with a vaccine particularly in the case of a poorly immunogenic antigen. Such a T cell helper peptide must be promiscuous in its ability to bind a broad range of MHC class II alleles due to broad allelic variation in the human population. We designed a chimeric MHC class II peptide (TpD) with epitopes from tetanus toxoid and diphtheria toxoid, separated by an internal cathepsin cleavage site. TpD was capable of inducing a memory recall response in peripheral blood mononuclear cells from 20/20 human donors. T cells responding to TpD showed a central memory phenotype. Immunization of mice with a synthetic nicotine nanoparticle vaccine containing TpD showed that the peptide was required for robust antibody production and resulted in a long term CD4 memory T cell recall response.