HIV-1 prophylactic vaccine comprised of topical DermaVir prime and protein boost elicits cellular immune responses and controls pathogenic R5 SHIV162P3

Topical DNA vaccination (DermaVir) facilitates antigen presentation to naive T cells. DermaVir immunization in mice, using HIV-1 Env and Gag, elicited cellular immune responses. Boosting with HIV-1 gp120 Env and p41 Gag augmented Th1 cytokine levels. Intramuscular DNA administration was less efficient in priming antigen-specific cytokine production and memory T cells. In rhesus macaques, DermaVir immunization induced Gag- and Env-specific Th1 and Th2 cytokines and generation of memory T cells. Boosting of DermaVir-primed serum antibody levels was noted following gp140(SHIV89.6P)/p27(SIV) immunization. Rectal challenge with pathogenic R5-tropic SHIV162P3 resulted in control of plasma viremia (4/5 animals) that was reflected in jejunum, colon and mesenteric lymph nodes. An inverse correlation was found between Gag- and Env-specific central memory T cell responses on the day of challenge and plasma viremia at set point. Overall, the topical DermaVir/protein vaccination yields central memory T cell responses and facilitates control of pathogenic SHIV infection.     

我国不同艾滋病候选疫苗小鼠细胞免疫应答评价

目的 比较我国不同艾滋病(AIDS)候选疫苗小鼠细胞免疫应答水平.方法 将我国6种艾滋病候选疫苗按其各自免疫程序,免疫BALB/c小鼠,分离脾细胞,利用酶联免疫斑点法(ELISPOT)与胞内因子染色法(ICS)检测其对HIV特异多肽的细胞免疫应答水平.结果 6种艾滋病候选疫苗利用潜在T细胞表位(PTE)Gag、Env和Pol肽库和ELISPOT法检测IFN-γ的分泌情况,结果各疫苗的小鼠细胞免疫阳转率为70%～100%,各疫苗引起特异细胞免疫应答的反应肽库不同,强度亦不相同.1#和2#疫苗利用ELISPOT检测分泌Th1类细胞因子IFN-γ和IL-2的小鼠脾细胞数量,两疫苗的IFN-γ和IL-2检测结果均呈中度相关性(r1=0.62,P1＜0.01;r2=0.79,P2＜0.01).ELISPOT和ICS检测1#疫苗免疫后分泌IFN-γ的小鼠脾细胞数,结果两种方法的小鼠阳转率均为66.7%(10/15),ELISPOT阳性细胞数与ICS阳性细胞比例呈中度相关(r=0.55,P＜0.05).结论 我国不同AIDS候选疫苗引起的小鼠细胞免疫应答在广度和强度上存在差别.ELISPOT或ICS检测IFN-γ等Th1类细胞因子可作为疫苗诱导小鼠细胞免疫应答的评价指标.     

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.     

RANTES和MIP-1α基因对HIV-1核酸疫苗诱导免疫应答的影响

目的　研究RANTES(活化T细胞调节的、正常T细胞表达和分泌的分子 )、MIP 1α(巨噬细胞炎症蛋白 1α)基因对HIV 1核酸疫苗诱导免疫应答的影响 ,以探求HIV 1核酸疫苗的新策略。方法　pCI neoGAG联合RANTES、MIP 1α基因或者pCI neoGAG单独免疫BALB c小鼠 ,采用ELISA检测免疫小鼠的特异性抗体和IFN γ水平 ,以MTT比色法检测免疫小鼠脾淋巴细胞的增殖 ,用乳酸脱氢酶 (LDH)试验检测小鼠特异性细胞毒性T淋巴细胞 (CTL)的应答。结果　与pCI neoGAG免疫组比较 ,pCI neoGAG联合RANTES、MIP基因免疫组小鼠血清的抗HIV 1p2 4抗体滴度升高 ,IFN γ升高 ,小鼠的脾淋巴细胞增殖实验刺激指数 (SI)以及特异性CTL活性均高 ,差异都有显著性 (P <0 .0 1)。结论　RANTES、MIP 1α基因联合HIV 1核酸疫苗免疫小鼠 ,可能增强特异性TH1细胞和CTL反应 ,RANTES、MIP 1α基因对体液免疫有加强作用。因此 ,RANTES、MIP 1α基因对于HIV 1核酸疫苗是具有较好应用前景的免疫佐剂。     

Studies on the cross-clade and cross-species conservation of HIV-1 Gag-specific CD8 and CD4 T cell responses elicited by a clade B DNA/MVA vaccine in macaques

Here, we evaluate the T cell responses raised by our HIV-1 clade B DNA/MVA vaccine for recognition of a HIV-1 circulating recombinant form (CRF) AG Gag sequence (CRF-02). The cross-clade activity for the AG sequence was better conserved for CD8 than CD4 T cells. CD8 T cells exhibited 75% conservation for height and 83% conservation for breadth, whereas CD4 responses exhibited 45% conservation for height and 50% conservation for breadth. Five CD8 epitopes and 8 CD4 epitopes were mapped. Three of the 5 CD8 epitopes and 2 of the 8 CD4 epitopes were conserved across multiple HIV-1 clades. Impressively, all of the CD8 epitopes and half of the CD4 epitopes have been reported for human infections. Our results demonstrate that the clade B DNA/MVA HIV vaccine elicits T cell responses against epitopes that are conserved in multiple clades and recognized by humans and macaques.     

Durable protection of rhesus macaques immunized with a replicating adenovirus-SIV multigene prime/protein boost vaccine regimen against a second SIVmac251 rectal challenge: role of SIV-specific CD8+ T cell responses

Previously, priming with replication-competent adenovirus-SIV multigenic vaccines and boosting with envelope subunits strongly protected 39% of rhesus macaques against rectal SIV(mac251) challenge. To evaluate protection durability, eleven of the protected and two SIV-infected unimmunized macaques that controlled viremia were re-challenged rectally with SIV(mac251). Strong protection was observed in 8/11 vaccinees, including two exhibiting <50 SIV RNA copies. Decreased viremia compared to na?ve controls was observed in the other three. The SIV-infected unimmunized macaques modestly controlled viremia but exhibited CD4 counts < or =200, unlike the protected macaques. Durable protection was associated with significantly increased SIV-specific ELISPOT responses and lymphoproliferative responses to p27 at re-challenge. After CD8 depletion, 2 of 8 re-challenged, protected vaccinees maintained <50 SIV RNA copies; SIV RNA emerged in 6. Re-appearance of CD8 cells and restoration of SIV-specific cellular immunity coincided with viremia suppression. Overall, cellular immunity induced by vaccination and/or low-level, inapparent viremia post-first SIV(mac251) challenge, was associated with durable protection against re-challenge.     

Strong cellular and humoral anti-HIV Env immune responses induced by a heterologous rhabdoviral prime-boost approach

Recombinant rhabdovirus vectors expressing human immunodeficiency virus (HIV) and/or simian immunodeficiency virus (SIV) proteins have been shown to induce strong immune responses in mice and rhesus macaques. However, the finding that such responses protect rhesus macaques from AIDS-like disease but not from infection indicates that further improvements for these vectors are needed. Here, we designed a prime-boost schedule consisting of a rabies virus (RV) vaccine strain and a recombinant vesicular stomatitis virus (VSV) both expressing HIV Envelope (Env). Mice were primed and boosted with the two vaccine vehicles by different routes and in different combinations. Mucosal and systemic humoral responses were assessed using enzyme linked immunosorbent assay (ELISA) while the cellular immune response was determined by an IFN-gamma ELISPOT assay. We found that an immunization combination of RV and VSV elicited the highest titers of anti-Env antibodies and the greatest amount of Env-specific IFN-gamma secreting cells pre- and post-challenge with a recombinant vaccinia virus expressing HIV(89.6) Env. Furthermore, intramuscular immunization did not induce antigen-specific mucosal antibodies while intranasal inoculation stimulated vector-specific IgA antibodies in vaginal washings and serum. Our results show that it is feasible to elicit robust cellular and humoral anti-HIV responses using two different live attenuated Rhabdovirus vectors to sequentially prime and boost.     

Long-term humoral and cellular immune responses elicited by a heterologous Plasmodium vivax apical membrane antigen 1 protein prime/adenovirus boost immunization protocol

Apical membrane antigen 1 (AMA-1) is an invasion-related Plasmodium antigen that is expressed during both intracellular and extracellular asexual stages of the parasite's life cycle, making it an ideal target for induction of humoral and cellular immune responses that can protect against malaria. We show here that when it is administered as a recombinant protein (P) in Montanide ISA720 adjuvant, followed by a recombinant human type 5 adenovirus (Ad), intense and long-lasting Plasmodium vivax AMA-1-specific antibody responses (including both IgG1 and IgG2a), as well as proliferative memory T cell responses, can be detected in immunized mice. Memory T cells displayed both central (CD44(hi) CD62L(hi)) and effector (CD44(hi) CD62L(lo)) phenotypes, with the central memory phenotype prevailing (56% of AMA-1-specific proliferating cells). Considering the main traits of the memory immune responses induced against AMA-1, this particular sequence of immunogens (P followed by Ad), but no others (Ad/Ad, Ad/P, or P/P), displayed an optimal synergistic effect. These results give further support to the need for preclinical studies of P. vivax vaccine candidate AMA-1 administered in prime/boost protocols that include recombinant proteins and adenoviral vectors.     

Expansion of restricted cellular immune responses to HIV-1 envelope by vaccination: IL-7 and IL-12 differentially augment cellular proliferative responses to HIV-1

The failure of immune effector mechanisms to control HIV-1 infection has important consequences for the human host. In a randomized cohort of HIV-infected patients, there was striking in vitro restriction of the proliferative response to HIV-1 envelope protein (Env), gp160; only 34% of patients recognized Env. Therapeutic vaccination with recombinant gp160 or gp120 (rgp160, rgp120) reversed the restriction in vitro, with Env recognition rising to 81%. Peripheral blood mononuclear cells (PBMC) from HIV-infected vaccine recipients, placebo recipients, and seronegative volunteers were cultured with exogenous IL-7 or IL-12 and either tetanus toxoid (TT) or gp160. IL-7 significantly augmented proliferative responses to TT and gp160, whereas IL-12 only affected proliferation to gp160. IL-7, but not IL-12, increased the number of HIV-infected placebo recipients who recognized rgp160. IL-12 had its greatest effect in the induction of rgp160-specific responses from seronegative individuals. The data suggest that these two cytokines have differential activity in the relief of restricted cellular immunity to Env; the predominant effect of IL-7 is in individuals who have been primed by exposure to antigen, while the effect of IL-12 is most evident in seronegative, unprimed individuals. Modification of restricted proliferative responses to Env by vaccination or cytokines in vitro suggests that strategies incorporating IL-7 or IL-12 as adjuvants may selectively boost cellular reactivity to HIV-1.     

Specific nature of cellular immune responses elicited by chimpanzees against HIV-1

Recent epidemiologic and phylogenetic analyses suggest that in the human population human immunodeficiency virus (HIV-1) is a relatively new pathogen that arose by zoonotic transmission from chimpanzees. In humans the morbidity and mortality figures due to HIV infection are extremely high. In a very small percentage of the human population, however, individuals have been identified who were infected for more than 20 years and have no evidence of disease progression. In contrast to most infected humans, almost all chimpanzees appear to be resistant to the pathologic effects caused by lentiviruses such as HIV-1. Here we review the characteristics of the HIV-1-specific cell-mediated immune responses mounted by chimpanzees, and we postulate the mechanisms that have evolved that facilitate their resistance to acquired immunodeficiency syndrome.     

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.     

HIV-1C亚型nef基因构建的DNA质粒诱导小鼠免疫反应的研究

目的构建表达中国流行株HIV-1C亚型调控nef基因的重组质粒pVAX-nef，并免疫BALB／c小鼠，观察其免疫效果，为探索新型HIV DNA疫苗提供数据。方法利用分子生物学技术，将nef基因克隆到pVAX，并在体外进行表达与鉴定。以纯化的Nef蛋白作为包被抗原，用ELISA检测其体液免疫反应，用ELISPOT检测其细胞免疫反应。结果重组质粒pVAX-nef成功构建。接种小鼠后2周，ELISPOT结果显示产生了针对HIV特异的CD4和CD8细胞抗原表位的免疫应答，且与免疫剂量存在一定的正相关性。ELISA实验诱导产生了抗HIV-1特异性抗体，其中40μg免疫组诱导的抗体水平最高。结论重组质粒pVAX-nef免疫小鼠后可有效地诱导机体产生细胞免疫和体液免疫反应。     

Enhanced humoral HIV-1-specific immune responses generated from recombinant rhabdoviral-based vaccine vectors co-expressing HIV-1 proteins and IL-2

Recombinant rabies virus (RV) vaccine strain-based vectors expressing HIV-1 antigens have been shown to induce strong and long-lasting cellular but modest humoral responses against the expressed antigens in mice. However, an effective vaccine against HIV-1 may require stronger responses, and the development of such an immune response may depend on the presence of certain cytokines at the time of the inoculation. Here, we describe several new RV-based vaccine vehicles expressing HIV-1 Gag or envelope (Env) and murine IL-2 or IL-4. Cells infected with recombinant RVs expressed high levels of functional IL-2 or IL-4 in culture supernatants in addition to HIV-1 proteins. The recombinant RV expressing IL-4 was highly attenuated in a cytokine-independent manner, indicating that the insertion of two foreign genes into the RV genome is mainly responsible for the attenuation observed. The expression of IL-4 resulted in a decrease in the cellular immune response against HIV-1 Gag and Env when compared with the parental virus not expressing IL-4 and only 2 of 20 mice seroconverted to HIV-1 Env after two inoculations. The IL-2-expressing RV was completely apathogenic after direct intracranial inoculation of mice. In addition, mice immunized with IL-2 maintained strong anti-HIV-1 Gag and Env cellular responses and consistently induced seroconversion against HIV-1 Env after two inoculations. This suggests the potential use of IL-2 in RV-based HIV-1 vaccine strategies, which may require the induction of both arms of the immune response.     

Breadth of cellular and humoral immune responses elicited in rhesus monkeys by multi-valent mosaic and consensus immunogens

To create an HIV-1 vaccine that generates sufficient breadth of immune recognition to protect against the genetically diverse forms of the circulating virus, we have been exploring vaccines based on consensus and mosaic protein designs. Increasing the valency of a mosaic immunogen cocktail increases epitope coverage but with diminishing returns, as increasingly rare epitopes are incorporated into the mosaic proteins. In this study we compared the immunogenicity of 2-valent and 3-valent HIV-1 envelope mosaic immunogens in rhesus monkeys. Immunizations with the 3-valent mosaic immunogens resulted in a modest increase in the breadth of vaccine-elicited T lymphocyte responses compared to the 2-valent mosaic immunogens. However, the 3-valent mosaic immunogens elicited significantly higher neutralizing responses to Tier 1 viruses than the 2-valent mosaic immunogens. These findings underscore the potential utility of polyvalent mosaic immunogens for eliciting both cellular and humoral immune responses to HIV-1.     

含密码子优化型HIV-1 gp120基因重组腺病毒的构建及其免疫效果研究

目的 构建能表达野生型和密码子优化型人免疫缺陷病毒Ⅰ型(HIV-1)B亚型中国流行株gp120基因的非复制型腺病毒。方法 按哺乳动物细胞偏好的密码子对HIV-1B亚型中国流行株Ch gp42的gp120基因进行优化，合成优化基因。将野生型和密码子优化的gp120基因插入穿梭质粒，再与腺病毒骨架质粒pAdEasy-1共转化E．coli BJ5183，获得重组子，转染293细胞后获得重组病毒。分别以两种重组腺病毒疫苗免疫小鼠，ELISA检测小鼠血清中的特异性抗体，乳酸脱氢酶法检测小鼠细胞毒性T淋巴细胞(CTL)反应。结果 获得两株重组腺病毒rAd-wt．gp120和rAd．mod．gp120，能正确表达Gp120。rAd-mod．gp120比rAd-wt．gp120蛋白表达水平明显提高。重组腺病毒免疫小鼠后能产生HIV-1特异性的抗体及CTL反应，rAd-mod．gp120组明显优于rAd-wt．gp120组。结论 成功构建了表达野生型和密码子优化的HIV-1 gp120基因的重组腺病毒，能诱导HIV-1特异性体液和细胞免疫反应。     

HIV-1 B''亚型gag-env融合基因的DNA疫苗构建和免疫原性分析

目的 构建HIV-1 B′亚型中国流行株gag和env融合基因的DNA疫苗，对其免疫原性进行研究。方法 根据已报道的HIV-1 B′亚型RIA2分离株gag和env基因的氨基酸序列按哺乳动物密码子使用频率进行优化并人工合成基因，插入真核表达载体pDRVISV1．0中，构建表达RL42 gag-env，融合蛋白的DNA疫苗，pSVRL／GE。用Western blot和抗gag p55抗体细胞内染色的方法体外检测pSVRUGE的表达效率。DNA疫苗pSVRL/GE免疫BALB／c小鼠后，用ELISPOT检测小鼠的细胞免疫反应。结果 限制性内切酶鉴定表明融合基因已成功插入pDRVISV1．0载体中，Western blot证实融合基因可有效表达融合蛋白；细胞内染色结果表明，pSVRL/GE转染的293T细胞中49．8％表达gag p55，荧光强度均值为924；而空载体pDRVISV1．0转染的293T细胞中非特异背景染色只有0．5％。经免疫的小鼠脾细胞体外用H-2^d限制性表位肽AMQMIKET刺激后，ELISPOT检测显示，pSVRUGE免疫小鼠每10^6脾细胞形成226个斑点（SD=140），而空载对照组每1驴脾细胞形成29个斑点（SD=16）（P〈0．05）。结论 所构建的DNA疫苗pSVRL/GE可高效表达相应抗原蛋白，并可有效激活机体的细胞免疫反应。     

沙眼衣原体主要外膜蛋白核酸疫苗诱导小鼠产生特异性细胞和体液免疫应答

目的构建沙眼衣原体主要外膜蛋白核酸疫苗,并观察其诱导小鼠产生的体液免疫和细胞免疫。方法将核酸疫苗(pcDNA3.1MOMP)或对照空质粒(pcDNA3.1)注射于4～6周龄小鼠后腿股四头肌,每次剂量为100mg。间隔2周加强免疫2次。末次免疫后,ELISA法测定脾淋巴细胞培养上清液中IFNγ及小鼠血清中抗MOMP水平;MTT法测定脾淋巴细胞特异性增殖反应。结果小鼠接种核酸疫苗后,能产生特异性抗体,第3次免疫后抗体最高滴度达1∶1024,培养上清液中IFNγ达(532.0±45.4)pg/mL;实验组小鼠脾淋巴细胞刺激指数为3.94±0.25,其抗原特异性反应明显高于对照组。结论沙眼衣原体主要外膜蛋白核酸疫苗能刺激机体产生特异的细胞免疫和体液免疫。     

Codelivery of a DNA vaccine and a protein vaccine with aluminum phosphate stimulates a potent and multivalent immune response

The study explores the possibility of efficiently codelivering DNA vaccines and protein-based vaccines by formulation with aluminum phosphate (AlPO₄). When mixed with aluminum adjuvants, plasmid DNA bound tightly onto aluminum hydroxide [Al(OH)₃] but not to AlPO₄. Different doses of DNA vaccines formulated with AlPO₄ [but not Al(OH)₃] induced enhanced humoral responses and supported priming of MHC class I restricted cellular immunity. Different proteins mixed with the plasmid DNA vaccine in the AlPO₄ formulation did not impair its immunogenicity. Coinjection of two different vaccine-relevant antigens in the same AlPO₄ formulation, one as a DNA vaccine and the other as a recombinant protein, elicited polyvalent, humoral, and cellular immune responses to all antigens delivered. The isotype profiles of the induced humoral responses and the cytokine profiles of the specifically primed T cell responses indicated that the combined vaccines supported copriming of Th1- and Th2-biased as well as balanced responses. These findings indicate that the AlPO₄ adjuvant, a widely accepted adjuvant in human vaccination practice, can be used to combine protein- and DNA-based vaccination to prime an enhanced and balanced specific immunity.Abbreviations CTL Cytotoxic T lymphocyte - HBcAg Hepatitis B core antigen - HBsAg Hepatitis B surface antigen - HEL Hen egg lysozyme - IFN Interferon - mAb Monoclonal antibody     

IL-12、IL-18基因对HIV-1外膜蛋白基因疫苗诱导的免疫应答的影响

目的 研究IL-12、IL-18基因对HIV-1外膜蛋白基因疫苗诱导免疫应答的影响，以探求HIV-1核酸疫苗的新策略。方法 pVAX1GP120联合IL-12、IL-18基因或者pVAX1GP120单独免疫BALB／c小鼠，采用ELISA检测免疫小鼠的特异性抗体和IFN-γ水平，以NIT比色法检测免疫小鼠脾淋巴细脯增殖，用乳酸脱氢酶（LDH）试验检测小鼠特异性细胞毒性T淋巴细胞（CIL）的应答。结果 与pVAX1GP120免疫组比较，pVAX1GP120联合IL-12、IL-18基因免疫组小鼠血清的抗HIV-1 gp120抗体滴度升高，IFN-γ升高，小鼠的脾淋巴细胞增殖实验刺激指数（SI）以及特异性CTL活性均高，差异均有统计学意义（P〈0．01）。结论 IL-12、IL-18基因联合HIV-1核酸疫苗免疫小鼠，有可能增强特异性TH1细胞和CTL反应，IL-12、IL-18基因对体液免疫可能有抑制作用。因此，IL-12、IL-18基因对于治疗性HIV-1核酸疫苗可能是具有较好应用前景的免疫佐剂。