Pre-clinical development of a multi-CTL epitope-based DNA prime MVA boost vaccine for AIDS

Reliable and effective methods for induction of cytotoxic T-lymphocytes (CTL) are constantly persued. Central to this search is work in animal models, which allow to test novel vaccine strategies and ultimately lead to a more efficient planning of clinical trials. Here, human immunodeficiency virus (HIV) vaccine candidates were constructed as a string of partially overlapping CTL epitopes (20 human, 3 macaque and 1 mouse) delivered and expressed using plasmid DNA and modified virus Ankara (MVA; an attenuated vaccinia virus), which are both vaccine vehicles acceptable for use in humans. In mice, these vaccines were shown to induce virus-specific interferon-gamma-producing and cytolytic CD8+ T-cells after a single intramuscular needle injection. When immunization protocols were sought which would improve the level of induced HIV-specific T-cells, DNA priming-MVA boosting was found to be the most potent protocol. The multi-epitope DNA also elicited CTL when delivered intradermally using the Accell gene delivery device (gene gun). Finally, a combined intradermal gene gun DNA-MVA vaccination regimen induced in macaques high frequencies of circulating CTL, which were comparable to those observed in simian immunodeficiency virus (SIV)-infected monkeys. Further optimization of this method in non-human primates is under way. Thus, a vaccination regimen for an effective elicitation of CTL has been developed which might facilitate evaluation of the role(s) that these lymphocytes play in the control of SIV and HIV infections.     

Protection against dengue virus by non-replicating and live attenuated vaccines used together in a prime boost vaccination strategy

A new vaccination strategy for dengue virus (DENV) was evaluated in rhesus macaques by priming with tetravalent purified inactivated virus (TPIV) or tetravalent plasmid DNA vaccines expressing the structural prME gene region (TDNA) then boosting 2 months later with a tetravalent live attenuated virus (TLAV) vaccine. Both vaccine combinations elicited virus neutralizing (N) antibodies. The TPIV/TLAV combination afforded complete protection against DENV 3 challenge at month 8. In a second experiment, priming with TPIV elicited N antibodies against all four serotypes (GMT 1:28 to 1:43). Boosting with TLAV led to an increase in the GMT for each serotype (1:500 to 1:1200 for DENVs 1, 3, and 4, and greater than 1:6000 for DENV 2), which declined by month 8 (GMT 1:62 for DENV 3, 1:154 for DENV 1, 1:174 for DENV 4, and 1:767 for DENV 2). After challenge with each one of the four DENV serotypes, vaccinated animals exhibited no viremia but showed anamnestic antibody responses to the challenge viruses.     

Preclinical evaluation of cellular immune responses elicited by a polyvalent DNA prime/protein boost HIV-1 vaccine

While DNA vaccines have been shown to prime cellular immune responses, levels are often low in nonhuman primates or humans. Hence, efforts have been directed toward boosting responses by combining DNA with different vaccination modalities. To this end, a polyvalent DNA prime/protein boost vaccine, consisting of codon optimized HIV-1 env (A, B, C, E) and gag (C) and homologous gp120 proteins in QS-21, was evaluated in rhesus macaques and BALB/c mice. Humoral and cellular responses, detected following DNA immunization, were increased following protein boost in macaques and mice. In dissecting cellular immune responses in mice, protein-enhanced responses were found to be mediated by CD4+ and CD8+ T cells with a Th1 cytokine bias. Our study reveals that, in addition to augmenting humoral responses, protein boosting of DNA-primed animals augments cellular immune responses mediated by CD8+ CTL, CD4+ T-helper cells and Th1 cytokines; thus, offering much promise in controlling HIV-1 in vaccinees.     

Flt3L及CCL5对prime/boost免疫策略中抗原特异性免疫应答的增强及抗肿瘤作用

目的:研究Flt3L与CCL5作为联合佐剂在prime/boost免疫策略中对HBc抗原特异性免疫应答的增强及抗肿瘤作用.方法:将两种细胞因子质粒与携带HBc抗原的DNA疫苗经肌内注射法共免疫小鼠, 免疫3次后再用原核表达的HBc颗粒蛋白或HBc DNA疫苗加强, 观察对稳定表达HBcAg 的小鼠黑色素瘤细胞(B16-HBc)的生长抑制作用;并分别采用MTT法检测荷瘤小鼠脾淋巴细胞增殖、流式细胞术检测脾CD8 T淋巴细胞中IFN-γ表达、ELISA法检测脾淋巴细胞培养上清IL-2、IL-4含量及乳酸脱氢酶(LDH)释放法检测特异性CTL杀伤活性.结果:与对照组相比, 佐剂联合DNA疫苗免疫经蛋白加强组(DDP/Adj)显著抑制肿瘤生长;佐剂联合DNA疫苗免疫组(DDD/Adj)及DDP/Adj组均可促进特异性淋巴细胞增殖反应(P＜0.05), 且DDP/Adj 高于DDD/Adj组(P＜0.05);DDD/Adj 及DDP/Adj组小鼠脾脏CD8 T淋巴细胞中IFN-γ表达、IL-2 表达水平及CTL杀靶活性均高于对照组(P＜0.01或P＜0.05), IL-4 表达水平在各组无显著区别(P＞0.05).结论:在prime/boost免疫策略中, 采用Flt3L与CCL5两种细胞因子联合应用可显著促进荷瘤小鼠产生抗原特异性免疫应答及抗肿瘤作用.     

DNA prime followed by protein boost enhances neutralization and Th1 type immunity against FMDV

Prime-boost strategy has been exhibited its potency to enhance immune responses, which would be important to the success to develop a vaccine against the foot-and-mouth disease virus (FMDV). An eukaryotic expression construct encoding the FMDV capsid VP1 protein with a recombinant VP1 protein or a commercial FMDV vaccine were tested in the prime-boost strategy in mice and cattle trials. The levels of induced specific antibodies, T cell proliferations, and DTH activities were significantly higher in the prime-boost groups than in those vaccinated with DNA, protein or FMDV vaccine alone. More importantly, the levels of neutralizing antibodies in the former groups were significantly higher than others and could last for at least four months in cattle trials. This study suggests that the prime-boost strategy significantly improves the effective immunity and may provide a longer protection against FMDV infection.     

A DNA prime-Mycobacterium bovis BCG boost vaccination strategy for cattle induces protection against bovine tuberculosis

The variable efficacy of bacillus Calmette-Guérin (Mycobacterium bovis BCG) in protecting humans and cattle against tuberculosis has prompted a search for a more effective vaccination regimen. A prime-boost strategy was investigated in cattle naturally sensitized to environmental mycobacteria by using a combination of three DNA vaccines coding for Hsp 65, Hsp 70, and Apa for priming, followed by a boost with BCG prior to experimental challenge with virulent M. bovis. Controls were vaccinated with DNA or BCG alone or were not vaccinated. The immune responses were monitored throughout the study, and protection was assessed based on reductions in the numbers of lesions and viable mycobacteria in lymph node samples. Vaccination with BCG alone or with a DNA prime-BCG boost regimen induced high levels of antigen-specific gamma interferon (IFN-gamma) in whole-blood cultures. In the prime-boost group there were fewer animals with severe lung lesions, fewer lymph nodes with lesions per animal, a smaller proportion of animals with lesions, lower mean lung and lymph node lesion scores, and less M. bovis isolated from retropharyngeal and thoracic lymph nodes compared to the results obtained for the nonvaccinated animals. The prime-boost regimen induced significant enhancement of protection in six parameters, compared with significant enhancement of protection in only two parameters for BCG alone. In addition, following challenge, in vitro IFN-gamma responses against ESAT-6 and CFP-10, as well as bovine tuberculin-induced skin test and in vitro IFN-gamma responses, were identified as immunological markers that predicted protection. The use of the prime-boost strategy suggested that a combination of vaccines may be better than a single vaccine for protection against tuberculosis.     

Robust immune response elicited by a novel and unique Mycobacterium tuberculosis protein using an optimized DNA/protein heterologous prime/boost protocol

An efficacious tuberculosis (TB) vaccine will probably need to induce both CD4 and CD8 T‐cell responses specific to a protective Mycobacterium tuberculosis antigen(s). To achieve this broad cellular immune response we tested a heterologous DNA/protein combination vaccine strategy. We used a purified recombinant protein preparation of a unique M. tuberculosis antigen (rMT1721) found in the urine of TB patients, an optimized plasmid DNA expressing this protein (DNA‐MT1721), and a Toll‐like receptor 4 agonist adjuvant. We found that priming mice with DNA‐MT1721 and subsequently boosting with rMT1721 elicited high titres of specific IgG1 and IgG2a antibodies as well as high magnitude and polyfunctional CD4⁺ T‐cell responses. However, no detectable CD8⁺ T‐cell response was observed using this regimen of immunization. In contrast, both CD4⁺ and CD8⁺ T‐cell responses were detected after a prime/boost vaccination regimen using rMT1721 as the priming antigen and DNA‐MT1721 as the boosting immunogen. These findings support the exploration of heterologous DNA/protein immunization strategies in vaccine development against TB and possibly other infectious diseases.     

CTLA4 targeting strategy in DNA vaccination against periodontitis

Periodontitis are among the most frequent infectious diseases affecting children and adolescents. The primary etiology of periodontal diseases is the bacterial plaque. Studies have demonstrated the feasibility of inducing immune responses by DNA vaccines against Porphyromonas gingivalis, the major aetiological agent of chronic periodontitis, and the subsequent development of periodontitis in animal models. But until now, the poor immunogenicity, particularly in large animals, is still a major problem in DNA vaccination. Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) is a membrane bound molecule mainly located on activated T cells. Its extracellular V-domain is considered to be involved in mediating the binding to the B7 molecule on antigen-presenting cells (APCs). By utilizing the interaction between CTLA4 and B7, specific antigens can be targeted to APCs by fusion to CTLA4 and DNA vaccines encoding CTLA4 and specific antigens greatly increased the systemic antibody responses following immunization in mice. We hypothesize that targeting antigens to APCs offers a potential strategy to enhance the immune responses generated by DNA vaccines against periodontitis.     

Comparison of single 1-day-old chick vaccination using a Newcastle disease virus vector with a prime/boost vaccination scheme against a highly pathogenic avian influenza H5N1 challenge

Avian influenza (AI) vaccines should be used as part of a whole comprehensive AI control programme. Vectored vaccines based on Newcastle disease virus (NDV) are very promising, but are so far licensed in only a few countries. In the present study, the immunogenicity and protection against a highly pathogenic H5N1 influenza challenge were evaluated after vaccination with an enterotropic NDV vector expressing an H5 haemagglutinin (rNDV-H5) in 1-day-old specific pathogen free chickens inoculated once, twice or once followed by a heterologous boost with an inactivated H5N9 vaccine (iH5N9). The heterologous prime/boost rNDV-H5/iH5N9 combination afforded the best level of protection against the H5N1 challenge performed at 6 weeks of age. Two rNDV-H5 administrations conferred a good level of protection after challenge, although only a cellular H5-specific response could be detected. Interestingly, a single administration of rNDV-H5 gave the same level of protection as the double administration but without any detectable H5-specific immune response. In contrast to AI immunity, a high humoral, mucosal and cellular NDV-specific immunity could be detected up to 6 weeks post vaccination after using the three different vaccination schedules. NDV-specific mucosal and cellular immune responses were slightly higher after double rNDV-H5 vaccination when compared with single inoculation. Finally, the heterologous prime/boost rNDV-H5/iH5N9 combination induced a broader detectable immunity including systemic, mucosal and cellular AI and NDV-specific responses.     

Production of antibodies to Little cherry virus 1 coat protein by DNA prime and protein boost immunization

Little cherry, an economically important disease of cherry is caused by at least two different viruses. One of these is Little cherry virus 1 (LChV-1) for the detection of which no efficient serological tools are available, so that diagnosis is based on molecular methods. In this study, different immunization strategies for producing antibodies against the viral coat protein of LChV-1 were tried, using either purified virus preparations, or bacterially expressed protein, or a DNA vector that expressed the cloned coat protein (CP) gene in vivo. Effective induction of specific antibodies to LChV-1 CP was obtained using DNA intramuscular immunization followed by a single boost with the recombinant protein. The entire coat protein sequence was cloned in a mammalian expression vector and, after being coated by an amphiphilic non-toxic reagent was delivered into rabbit. A protein boost increased the specific immune response against the virus protein. The sensitivity of this antiserum is lower if compared with that of antisera raised conventionally against other viruses, thus it requires improvements for use for diagnostic purposes.     

ELISPOT analysis of a new CTL based DNA vaccine for HIV-1 using GM-CSF in DNA prime/peptide boost strategy: GM-CSF induced long-lived memory responses

Genetic adjuvants have potential role in improvement of immune responses against DNA vaccines. GM-CSF as a genetic adjuvant can recruit and augment dendritic cell numbers in the site of immune responses and thereby induce cellular and humoral immune responses. Here we show that co-immunization of a DNA vaccine from HIV-1P24-Nef with GM-CSF in DNA priming and peptide boost strategy increases the immunogenicity of our candidate vaccine. Analysis of immune response shows that co-immunization with GM-CSF boosts cellular immune responses through increasing proliferation activity and CTL function. Results of cytokine profile studies show that both IL-4 and IFN-γ levels were augmented. Also, co-immunization with GM-CSF resulted in a higher level of total IgG, comprising approximately equal levels of both specific IgG1 and IgG2a subtypes. Monitoring of cellular and humoral immune responses for 20 weeks after final immunization revealed the aptitude of GM-CSF for inducing long-lived humoral and cell mediated immune responses. Overall, our results suggest that GM-CSF is able to induce long term memory for the HIV-1 P24-Nef vaccine candidate while the exact mechanisms involved remained to be clarified.     

Design and evaluation of antigen-specific vaccination strategies against cancer

After studies in preclinical mouse models, the efficacy and safety of tumor-specific vaccination strategies is currently being evaluated in cancer patients. The first wave of clinical trials has shown that in general such vaccination strategies are safe. However examples of clinical responses, especially in conjunction with vaccine-induced immune responses, are still scarce. The fact that most trials have so far been performed with end-stage cancer patients can largely account for this deficit. Greater efficacy of anticancer vaccines is expected in patients with less-progressed disease. In addition, the detection of both natural and vaccine-induced T cell immunity needs further improvement.     

Immunization of rhesus macaques with a polyvalent DNA prime/protein boost human immunodeficiency virus type 1 vaccine elicits protective antibody response against simian human immunodeficiency virus of R5 phenotype

The immunogenicity of a poylvalent HIV-1 vaccine comprised of Env antigens from primary R5 isolates was evaluated in rhesus macaques. DNA vaccines encoding four Env antigens from multiple HIV-1 subtypes and HIV-1 Gag antigen from a single subtype elicited a persistent level of binding antibodies to gp120 from multiple HIV-1 isolates that were markedly enhanced following boosting with homologous gp120 proteins in QS-21 adjuvant irrespective of the route of DNA immunization. These sera neutralized homologous and, to a lesser degree, heterologous HIV-1 isolates. Four of the six immunized animals were completely protected following rectal challenge with a SHIV encoding Env from HIV-1(Ba-L), whereas the virus load was reduced in the remaining animals compared to na?ve controls. Hence priming with DNA encoding Env antigens from multiple HIV-1 clades followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of protecting macaques against mucosal transmission of R5 tropic SHIV isolate.     

癌胚抗原阴性的结、直肠癌检测和结直肠癌预后相关生物标记

目的探讨结直肠癌的发生、发展过程中患者血清质谱多肽蛋白图谱的变化,筛选与结直肠癌预后及癌胚抗原(CEA)阴性检测相关的肿瘤标记分子。方法用蛋白指纹图谱技术检测结直肠癌,结直肠管状腺瘤患者和健康者血清质谱多肽蛋白图谱。结果初步筛选出对结直肠癌有代表性的7个差异蛋白;2个与其淋巴结转移相关的差异蛋白;4个与其远处转移相关的差异蛋白;3个在其根治性切除后表达下降的差异蛋白;而由3398·3u、5477·1u和8453·9u组成的诊断模型对CEA阴性表达结直肠癌的阳性检测率为100%。结论蛋白指纹图谱技术可筛选出有意义的生物标记差异蛋白,这对结直肠癌预后判断、CEA阴性的结直肠癌检测和改变结直肠癌的进程具有重要意义。     

DNA vaccination against tuberculosis: expression of a ubiquitin-conjugated tuberculosis protein enhances antimycobacterial immunity

Genetic immunization is a promising new technology for developing vaccines against tuberculosis that are more effective. In the present study, we evaluated the effects of intracellular turnover of antigens expressed by DNA vaccines on the immune response induced by these vaccines in a mouse model of pulmonary tuberculosis. The mycobacterial culture filtrate protein MPT64 was expressed as a chimeric protein fused to one of three variants of the ubiquitin protein (UbG, UbA, and UbGR) known to differentially affect the intracellular processing of the coexpressed antigens. Immunoblot analysis of cell lysates of in vitro-transfected cells showed substantial differences in the degradation rate of ubiquinated MPT64 (i.e., UbG64 < UbA64 < UbGR64). The specific immune response generated in mice correlated with the stability of the ubiquitin-conjugated antigen. The UbA64 DNA vaccine induced a weak humoral response compared to UbG64, and a mixed population of interleukin-4 (IL-4)- and gamma interferon (IFN-gamma)-secreting cells. Vaccination with the UbGR64 plasmid generated a strong Th1 cell response (high IFN-gamma, low IL-4) in the absence of a detectable humoral response. Aerogenic challenge of vaccinated mice with Mycobacterium tuberculosis indicated that immunization with both the UbA64- and UbGR64-expressing plasmids evoked an enhanced protective response compared to the vector control. The expression of mycobacterial antigens from DNA vaccines as fusion proteins with a destabilizing ubiquitin molecule (UbA or UbGR) shifted the host response toward a stronger Th1-type immunity which was characterized by low specific antibody levels, high numbers of IFN-gamma-secreting cells, and significant resistance to a tuberculous challenge.     

Dendritic/tumor fusion cell-based vaccination against cancer

A promising area of investigation is the use of cancer vaccines to eliminate residual tumor cells. Dendritic cells (DCs) are potent professional antigen-presenting cells able to induce primary immune responses. DCs capture and process antigens into peptides and present them to T cells and B cells through MHC class I and II molecules. An alternative approach to the induction of antitumor immunity is the use of fusions of DCs and tumor cells. In this approach, a broad spectrum of tumor-associated antigens, including those known and unidentified, are processed endogenously and presented by MHC class I and II pathways in the context of costimulatory signals. In animal studies, vaccination with DC/tumor fusion cells results in the elimination of established lung metastasis. Preclinical human studies have demonstrated that DC/tumor fusion cells induce antigen-specific polyclonal cytotoxic T-lymphocyte responses against autologous tumor in vitro. In clinical studies, vaccination of cancer patients with autologous DC/tumor fusion cells is associated with immunological and clinical responses in a subset of patients. Future studies should be investigated to improve the immunogenicity of DC/tumor fusion cell preparations. This review provides a general overview of the DC/tumor fusion cell-based vaccine and summarizes some of the recent advances in this field.     

Enhancing Th2 immune responses against amyloid protein by a DNA prime-adenovirus boost regimen for Alzheimer's disease

Accumulation of aggregated amyloid beta-protein (Abeta) in the brain is thought to be the initiating event leading to neurodegeneration and dementia in Alzheimer's disease (AD). Therefore, therapeutic strategies that clear accumulated Abeta and/or prevent Abeta production and its aggregation are predicted to be effective against AD. Immunization of AD mouse models with synthetic Abeta prevented or reduced Abeta load in the brain and ameliorated their memory and learning deficits. The clinical trials of Abeta immunization elicited immune responses in only 20% of AD patients and caused T-lymphocyte meningoencephalitis in 6% of AD patients. In attempting to develop safer vaccines, we previously demonstrated that an adenovirus vector, AdPEDI-(Abeta1-6)11, which encodes 11 tandem repeats of Abeta1-6 can induce anti-inflammatory Th2 immune responses in mice. Here, we investigated whether a DNA prime-adenovirus boost regimen could elicit a more robust Th2 response using AdPEDI-(Abeta1-6)11 and a DNA plasmid encoding the same antigen. All mice (n=7) subjected to the DNA prime-adenovirus boost regimen were positive for anti-Abeta antibody, while, out of 7 mice immunized with only AdPEDI-(Abeta1-6)11, four mice developed anti-Abeta antibody. Anti-Abeta titers were indiscernible in mice (n=7) vaccinated with only DNA plasmid. The mean anti-Abeta titer induced by the DNA prime-adenovirus boost regimen was approximately 7-fold greater than that by AdPEDI-(Abeta1-6)11 alone. Furthermore, anti-Abeta antibodies induced by the DNA prime-adenovirus boost regimen were predominantly of the IgG1 isotype. These results indicate that the DNA prime-adenovirus boost regimen can enhance Th2-biased responses with AdPEDI-(Abeta1-6)11 in mice and suggest that heterologous prime-boost strategies may make AD immunotherapy more effective in reducing accumulated Abeta.     

结核分枝杆菌联合DNA疫苗初免-BCG加强的免疫效果观察

目的:研究并比较结核分枝杆菌免疫保护性抗原DNA(Ag85A和ESAT-6)疫苗联合免疫,BCG免疫以及联合DNA疫苗初免-BCG加强免疫等不同的免疫策略,诱导免疫应答效果观察.方法:健康雌性BALB/c小鼠24只,随机分成PBS 阴性对照组,DNA初免-BCG异源加强组,DNA(Ag85A和ESAT-6)初免DNA同源加强组和BCG阳性对照组,共进行3次免疫,初免2次,最后1次加强,间隔2周1次.PBS组3次均注射PBS 溶液;DNA/BCG组以质粒DNA免疫2次,最后1次以BCG加强免疫;DNA/DNA组3次均以质粒DNA进行免疫;BCG组则注射PBS溶液2次后以BCG免疫.末次免疫后4、6、8周分别分离血清测定总IgG水平,同时分离小鼠脾细胞,体外经TB-PPD刺激后进行淋巴细胞增殖实验(XTT法)并测定脾细胞培养上清中IFN-γ和IL-4水平.结果:DNA/BCG、DNA/DNA、BCG组体外经TB-PPD刺激后均检测到特异性IgG抗体产生,3组平均效价为1:120、1:160、1:80,DNA/DNA组的抗体效价高于另外2组;小鼠脾细胞体外经TB-PPD刺激后,均能产生特异性淋巴细胞增殖并诱生较强的IFN-γ反应,其中DNA/BCG组IFN-γ的分泌水平高于DNA/DNA组和BCG组(P<0.05).结论:联合DNA疫苗初免-BCG加强的免疫策略能在小鼠体内诱导较强的特异性细胞免疫反应,产生高水平的IFN-γ.     

水痘疫苗应急接种效果评价

目的 评价水痘疫苗应急接种效果及暴发后人群抗体水平,指导水痘暴发疫情控制.方法 以2004-2006年发生水痘暴发的17所学校为研究对象,对其中5所学校的患者及密切接触者进行问卷调查,并采集2所学校水痘疫苗应急接种后应急接种者免疫前后血清标本各85份进行水痘疫苗血清抗体滴度测定.结果 应急接种前学校水痘罹患率为6.22％,应急接种后学校水痘罹患率为1.65％,两者的差别有统计学意义(x2=10.99,P＜0.01);接种疫苗者与未接种水痘者的水痘罹患率分别为8.60％和19.89％,免疫人群的水痘罹患率明显低于未免人群(x2=20.70,P＜0.01);接种水痘疫苗后血清抗体有效率为90.70％,抗体阳转率为87.80％,免疫成功率为70.70％.结论 应急接种对控制学校水痘疫情暴发有重要作用,建议在出现水痘病例后,依据病例数目和波及范围,进行相应范围内的应急接种.