增殖型腺病毒的改良及应用

增殖型腺病毒能在肿瘤细胞中复制并裂解肿瘤细胞,释放出的子代病毒再感染邻近肿瘤细胞直至完全杀灭肿瘤,却不影响正常细胞的功能。同时,增殖型腺病毒还是一种有效的基因治疗载体,可通过病毒自身增殖提高目的基因的拷贝数,从而更高效率地表达外源性治疗基因,增强抗肿瘤效应。本文着重介绍增殖型腺病毒载体改良和应用的最新进展,并对其研究前景进行展望,以期对增殖型腺病毒的发展有所帮助。     

Development of a Serum-free Suspension Process for the Production of a Conditionally Replicating Adenovirus using A549 Cells

Conditionally replicating adenoviruses (CRAVs) are a group of recombinant human adenoviruses genetically engineered to replicate in selected tissues, such as tumors. These viruses could potentially offer significant medicinal benefits, since the restrictive replication of these viral vectors leads to the lysis of target cells without harm to the surrounding tissues. The in vitro propagation and amplification of the CRAV vectors often requires special host cells with deregulated growth control pathways. In order to develop an efficient cell culture process for the scaleable production of a CRAV vector, A549 cells, a human lung carcinoma cell line normally cultured in adherent culture, were adapted to suspension culture. CRAV production was demonstrated with the suspension-adapted A549 cells and a baseline production process was developed in shake flasks. The ability to scale-up virus production was confirmed in stirred tank bioreactors. Molecular characterization of the suspension-adapted A549 cells indicates no significant changes in cellular mechanisms related to adenovirus infection.     

Enhanced Expression of Adenovirus Encoding rhEPO Assisted by BAPTA

Infection efficiency is the key issue for gene delivery using adenovirus vector and usually unsatisfactory. In this study, recombinant adenoviruses encoding recombinant human EPO were prepared using the Adeasy system, and injected into the mammary gland of goats via the teat canal. BAPTA was used to treat the mammary gland to facilitate adenoviruses infection compared with EGTA. Milk serum was collected from the infected mammary gland and characterized by ELISAs and Western blotting. Expression level of rhEPO from the group treated by BAPTA was higher than that treated by EGTA.     

A Mouse Model of Adoptive Immunotherapeutic Targeting of Autoimmune Arthritis Using Allo-Tolerogenic Dendritic Cells

Objective

Tolerogenic dendritic cells (tDCs) are immunosuppressive cells with potent tolerogenic ability and are promising immunotherapeutic tools for treating rheumatoid arthritis (RA). However, it is currently unknown whether allogeneic tDCs (allo-tDCs) induce tolerance in RA, and whether the numbers of adoptively transferred allo-tDCs, or the requirement for pulsing with relevant auto-antigens are important.

Methods

tDCs were derived from bone marrow precursors of C57BL/B6 mice, which were induced in vitro by GM-CSF, IL-10 and TGF-β1. Collagen-induced arthritis (CIA) was modeled in D1 mice by immunization with type II collagen (CII) to test the therapeutic ability of allo-tDCs against CIA. Clinical and histopathologic scores, arthritic incidence, cytokine and anti-CII antibody secretion, and CD4⁺Th subsets were analyzed.

Results

tDCs were characterized in vitro by a stable immature phonotype and a potent immunosuppressive ability. Following adoptive transfer of low doses (5×10⁵) of CII-loaded allo-tDCs, a remarkable anti-arthritic activity, improved clinical scores and histological end-points were found. Serological levels of inflammatory cytokines and anti-CII antibodies were also significantly lower in CIA mice treated with CII-pulsed allo-tDCs as compared with allo-tDCs. Moreover, treatment with allo-tDCs altered the proportion of Treg/Th17 cells.

Conclusion

These findings suggested that allo-tDCs, especially following antigen loading, reduced the severity of CIA in a dose-dependent manner. The dampening of CIA was associated with modulated cytokine secretion, Treg/Th17 polarization and inhibition of anti-CII secretion. This study highlights the potential therapeutic utility of allo-tDCs in autoimmune arthritis and should facilitate the future design of allo-tDC immunotherapeutic strategies against RA.     

Enhanced Expression of miR-425 Promotes Esophageal Squamous Cell Carcinoma Tumorigenesis by Targeting SMAD2

Esophageal squamous cell carcinoma(ESCC) is one of the most common and deadly cancers in the world. Currently, clinical therapy of ESCC remains limited and the five-year survival rate is poor. The function of miR-425 has been reported in multiple human cancers.However, the tumorigenic role and clinical significance of miR-425 in ESCC remains unclear. We found that enhanced expression of miR-425 in ESCC cell lines not only promoted cell proliferation and colony formation, but also increased cellular metastasis. Furthermore, we revealed the mechanism that miR-425 inhibited the expression of SMAD2 by targeting the second binding site in the 30-untranslated region(30-UTR) in ESCC. This mode of action influenced not only SMAD2 mRNA expression but also protein expression. In addition, we detected the expression of miR-425 in ESCC tissues and plasma. Moreover, we analyzed the relationship between miR-425 expression and SMAD2 m RNA expression. We found that miR-425 was overexpressed in ESCC tissues and the plasma relative to adjacent normal tissues and plasma of healthy individuals. Furthermore, there was a negative correlation between miR-425 expression and SMAD2. Taken together, our results show that miR-425 functions as an oncogene by targeting the 30-UTR of SMAD2 and indicate the potential utility of plasma miR-425 as a novel biomarker for ESCC diagnosis.     

7型腺病毒E1A基因克隆及真核表达

为了研究7型腺病毒(Ad7)E1A在感染中的致病机制,分离了Ad7d2病毒株,构建了Ad7 E1A基因的真核表达体系.用A549细胞分离培养痰液标本中的腺病毒,应用重叠延伸PCR扩增Ad7 E1A基因外显子,产物克隆至真核表达载体pIRES-Neo,构建重组子pIRES-Neo-Ad7E1A并转染A549细胞,利用Western印迹对其表达产物进行鉴定.克隆测序显示扩增的Ad7 E1A基因外显子包含了完整的编码区基因,pIRES-Neo-Ad7E1A转染A549细胞的表达产物经Western印迹鉴定与设计相符.成功建立了Ad7 E1A 基因的真核表达体系.     

Enhanced Post-Irradiation Recovery of the Haemopoietic System In Animals Pretreated With A Variety of Cytotoxic Agents

It is known that pretreatment of mice with bacterial endotoxin and certain stathmokinetic agents between 1 and 3 days prior to exposure to ionizing radiation reduce radiation lethality. In this communication it is shown that pretreatment with cytosine arabinoside, methotrexate, nortestosterone and chlorambucil reduces radiation (1000 rad) induced lethality. This reduction can be ascribed to enhanced regeneration of the haemopoietic system in pretreated animals and not to increased survival of colony-forming cells (CFU) in these animals. Regeneration of CFUs was underway within 24 hr after 900 rad in the pretreated mice but did not start until day 3 in mice treated with γ radiation only. Two agents, namely radiation itself (either 75 or 150 rad) and busulphan (10 mg/kg) did not reduce the lethal effects of subsequent γ irradiation nor enhance the regeneration of CFUs, even though radiation, like the protective cytosine arabinoside, induces early CFUs proliferation. The administration of nucleoside precursors of DNA enhanced regrowth of haemopoietic stem cells to an extent comparable with that of the most effective pretreatment, cytosine arabinoside. It is postulated that drugs like cytosine arabinoside operate by causing cell death, providing a source of DNA that can enhance the regrowth of surviving stem cells in the bone marrow.     

顺铂靶向食管癌细胞周期Cx43表达的研究

目的:为研究顺铂治疗食管鳞癌细胞的靶向作用。方法:本研究使用流式细胞技术双变量分析检测顺铂对食管癌细胞周期进程和癌细胞周期的连接蛋白43(connexin 43,Cx43)表达的影响。结果:顺铂对食管鳞癌细胞周期的影响主要作用于S期的DNA复制,细胞阻滞于S期,G2/M期减少。顺铂诱导食管鳞癌细胞周期S和G2/M期的Cx43表达的大幅度改变。低浓度顺铂(由0~2μmol/L),Cx43表达增强;顺铂渐高浓度(2~12μmol/L),细胞Cx43表达由强逐渐变弱,特别是G2/M期细胞的Cx43表达活跃,易受顺铂影响。结论:我们的研究表明以顺铂处理食管鳞癌细胞,癌细胞周期的S期和G2/M期的Cx43表达与S期的DNA复制一样可作为的潜在治疗靶标。顺铂靶向作用细胞周期S和/或G2/M期细胞的特性可能减少或避免对非分裂细胞的影响。     

人CUIAA重组腺病毒载体的构建及其在PC-12细胞中的表达特点

目的构建并鉴定带有绿色荧光蛋白（green fluorescence protein,GFP）报告基因的人源CUL4A（hCUIAA）基因腺病毒表达载体Ad—hCUIAA—GFP,探求bCUIAA在PC-12细胞中的表达特点。方法扩增hCUIAA基因,并通过In—FusionPCR克隆技术构建穿梭质粒pDC315-EGFP—hCUIAA,利用AdMaxTM腺病毒包装系统将该穿梭质粒与腺病毒表达载体骨架质粒pBHGloxAEl,E3Cre共转染HEK293细胞,经GFP荧光检测和Western印迹检测确认hCUIAA的表达后,进一步通过病毒扩增及纯化得到hCUL4A重组腺病毒载体Ad—hCUIAA—GFP。将该载体转染Pc—12细胞,观察hCUIAA—GFP融合蛋白在Pc-12细胞中的表达情况。结果成功获得了较高滴度的腺病毒载体Ad—hCUIAA—GFP（1．6×10^12pfu／m1）。荧光检测表明,Ad—hCUIAA—GFP转染Pc-12细胞后72h内,病毒转染率随着时间和病毒转染滴度的增加而增加。DAPI细胞核荧光染色结果表明,hCUIAA—GFP的表达主要集中在细胞质部分。GFP荧光检测及Western印迹检测结果显示,hCUIAA—GFP在Pc-12细胞中的表达量随时间和病毒转染滴度的增加而增加。结论带GFP的hCUIAA重组腺病毒载体Ad—hCUIAA—GFP构建成功,掌握了其转染Pc-12细胞的最佳滴度及其在Pc-12细胞中的时空表达特点,为今后对hCUIAA在PC-12细胞中的功能性研究奠定了基础。     

A Shrimp C-type Lectin Inhibits Proliferation of the Hemolymph Microbiota by Maintaining the Expression of Antimicrobial Peptides

Some aquatic invertebrates such as shrimp contain low albeit stable numbers of bacteria in the circulating hemolymph. The proliferation of this hemolymph microbiota in such a nutrient-rich environment is tightly controlled in healthy animals, but the mechanisms responsible had remained elusive. In the present study, we report a C-type lectin (MjHeCL) from the kuruma shrimp (Marsupenaeus japonicus) that participates in restraining the hemolymph microbiota. Although the expression of MjHeCL did not seem to be modulated by bacterial challenge, the down-regulation of its expression by RNA interference led to proliferation of the hemolymph microbiota, ultimately resulting in shrimp death. This phenotype was rescued by the injection of recombinant MjHeCL, which restored the healthy status of the knockdown shrimp. A mechanistic analysis revealed that MjHeCL inhibited bacterial proliferation by modulating the expression of antimicrobial peptides. The key function of MjHeCL in the shrimp immune homeostasis might be related to its broader recognition spectrum of the hemolymph microbiota components than other lectins. Our study demonstrates the role of MjHeCL in maintaining the healthy status of shrimp and provides new insight into the biological significance of C-type lectins, a diversified and abundant lectin family in invertebrate species.     

荷载金黄色葡萄球菌肠毒素A 基因的双调控溶瘤腺病毒载体 的构建、鉴定及其抗膀胱肿瘤作用

目的:构建表达超抗原金黄色葡萄球菌肠毒素A基因的双调控选择增殖型溶瘤腺病毒SG502-SEA及对照组携带超抗原SEA基因的非增殖溶瘤腺病毒DC318-SEA,并探讨其潜在的抗膀胱肿瘤作用.方法:将超抗原SEA基因片段经SpeⅠ和SalⅠ双酶切后,克隆入非增殖溶瘤腺病毒载体pSG218中,将鉴定正确的腺病毒载体命名为pDC318-SEA.同样方法将超抗原SEA基因片段克隆入双调控特异性增殖溶瘤腺病毒载体pSG502中,将鉴定正确的腺病毒载体命名为pSG502-SEA.将以上两种携带SEA基因的病毒载体与病毒骨架质粒PPE3-ccdB共转染293细胞,9～14d出现病毒空斑,经过3次病毒空斑纯化,提取腺病毒DNA,应用PCR进行鉴定,经鉴定正确的腺病毒分别命名为DC318 - SEA和SG502-SEA.大量扩增后,氯化铯梯度离心纯化腺病毒,测病毒滴度.结果:经PCR及酶切鉴定,SEA基因成功克隆到两病毒载体中,可以表达SEA基因,且病毒滴度为2.5×101pfu/ml.结论:成功构建表达超抗原SEA基因的双调控增殖型溶瘤腺病毒SG502-SEA及对照组携带超抗原SEA基因的非增殖溶瘤腺病毒DC318-SEA,为下一步抗膀胱肿瘤体内外实验奠定基础.     

A Novel Bicistronic High-Capacity Gutless Adenovirus Vector That Drives Constitutive Expression of Herpes Simplex Virus Type 1 Thymidine Kinase and Tet-Inducible Expression of Flt3L for Glioma Therapeutics

Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Conditional cytotoxic/immune-stimulatory gene therapy (Ad-TK and Ad-Flt3L) elicits tumor regression and immunological memory in rodent GBM models. Since the majority of patients enrolled in clinical trials would exhibit adenovirus immunity, which could curtail transgene expression and therapeutic efficacy, we used high-capacity adenovirus vectors (HC-Ads) as a gene delivery platform. Herein, we describe for the first time a novel bicistronic HC-Ad driving constitutive expression of herpes simplex virus type 1 thymidine kinase (HSV1-TK) and inducible Tet-mediated expression of Flt3L within a single-vector platform. We achieved anti-GBM therapeutic efficacy with no overt toxicities using this bicistronic HC-Ad even in the presence of systemic Ad immunity. The bicistronic HC-Ad-TK/TetOn-Flt3L was delivered into intracranial gliomas in rats. Survival, vector biodistribution, neuropathology, systemic toxicity, and neurobehavioral deficits were assessed for up to 1 year posttreatment. Therapeutic efficacy was also assessed in animals preimmunized against Ads. We demonstrate therapeutic efficacy, with vector genomes being restricted to the brain injection site and an absence of overt toxicities. Importantly, antiadenoviral immunity did not inhibit therapeutic efficacy. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L genes. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, affecting ∼18,000 new patients every year; its prognosis remains poor despite standard treatment with surgery, radiotherapy, and chemotherapy (temozolomide) (36-38, 44; J. C. Buckner, presented at the ASCO Annual Meeting, 2009). Complete resection is mostly impossible due to the highly infiltrative nature of this disease. Residual GBM cells remaining within the nonneoplastic brain parenchyma eventually lead to tumor recurrence that is resistant to conventional chemotherapy and radiotherapy, ultimately leading to the patient''s death (44). Several dendritic cell vaccination strategies aiming to stimulate the patient''s immune system to seek out and destroy residual brain tumor cells are currently under preclinical and clinical development and constitute a promising adjuvant treatment for GBM (18, 24, 42, 43, 45).We have developed a novel immunotherapeutic approach for GBM using first-generation adenoviral vectors (Ads) to deliver a combination of therapeutic transgenes into the tumor mass (2, 7, 9, 12, 14, 17, 48) which is slated to begin phase I clinical testing this year. In our strategy, rather than vaccinating against tumor antigens, we aim to reconstruct an immune circuit that is absent from the normal brain. Our gene therapy strategy consists of the conditionally cytotoxic herpes simplex virus type 1 thymidine kinase (TK) (2, 12), which kills proliferating tumor cells in the presence of the prodrug ganciclovir (GCV), used in combination with human soluble Fms-like tyrosine kinase 3 ligand (Flt3L), which recruits bone marrow-derived dendritic cells (DCs) to the normal brain or brain tumor microenvironment in mice (11, 12) and rats (1, 13). We previously showed that codelivery of Ad-Flt3L and Ad-TK into the brain tumor milieu induces an anti-GBM-specific immune response (9, 12, 17, 27, 48), leading to long-term survival of rats bearing intracranial CNS1, 9L, and F98 tumors (2, 17, 27) and mice bearing intracranial GL26, GL261, and B16-F10 tumors (12, 48). In addition, the combination of Ad-Flt3L plus Ad-TK induces GBM-specific immunological memory that improves survival in intracranial multifocal and recurrent models of GBM in both rats and mice (9, 12, 17, 20, 22, 27).First-generation adenoviral vectors have been used in clinical trials to deliver HSV1-TK (31). Ad-TK delivered into the margins of the tumor cavity after surgical resection of GBM was well tolerated in over 70 patients in six early clinical trials (19, 34). Publication of final results from a large, multicenter phase III trial involving 251 patients is awaited; preliminary analysis indicates small but statistically significant benefits from adding gene therapy to standard care. Final analysis will have to wait for the detailed publications (3, 16).To provide stable, long-term therapeutic transgene expression, “gutless,” high-capacity adenoviral (HC-Ad) vectors have been engineered in which all viral encoding genes have been eliminated and replaced with inert, noncoding stuffer DNA sequences (26, 30). In the presence of an anti-adenoviral immune response, transgene expression in the brain from first-generation Ads is reduced, while expression from HC-Ad vectors remains stable for at least 1 year (4, 25, 28). Therefore, we engineered two HC-Ad vectors to either constitutively express TK (HC-Ad-TK) (21, 27) or express Flt3L under the control of the tightly regulatable mCMV-TetOn expression system (HC-Ad-TetOn-Flt3L) (6, 8, 27, 47). Intratumoral administration of HC-Ad-TK and HC-Ad-TetOn-Flt3L led to high levels of therapeutic efficacy, and the therapy was well tolerated, with no overt toxicity and no spread of vector genomes outside the injected brain hemisphere in treated Lewis rats bearing syngeneic intracranial brain tumors (27).To reduce vector dose and facilitate GMP manufacturing of the clinical product, we engineered a novel, bicistronic HC-Ad vector that encodes, for the first time, both constitutively expressed HSV1-TK and inducible Flt3L from a single HC-Ad vector genome. Herein we demonstrate therapeutic efficacy of the bicistronic HC-Ad in naïve rats bearing intracranial tumors and also in tumor-bearing rats which had been exposed to adenoviruses and therefore exhibited anti-Ad immunity. The safety profile of this approach was assessed by evaluating the biodistribution of HC-Ad vector genomes, systemic and neurological toxicity, and behavioral abnormalities over the course of 1 year posttreatment. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.     

Design and Expression of Polymeric Immunoglobulin Fusion Proteins: A Strategy for Targeting Low-Affinity Fcγ Receptors

We have developed a family of cloning vectors that direct expression of fusion proteins that mimic aggregated immunoglobulin (IgG) (AIG) and immune complex function with respect to their interactions with FcγR and that allow for the inclusion and targeting of a second protein domain to cells expressing FcγR. This was accomplished by expressing multiple linear copies of the hinge and CH2 domains (HCH2) of human IgG₁ fused to the framework region of human IgG₁ . Convenient restriction sites allow for the facile introduction of additional amino-terminal domains. The resulting molecule is tripartite. The carboxyl-IgG₁ framework domain provides stability and permits dimerization, and the intervening polymer provides increased effector function and targeting to FcγR while the amino-terminal domain can deliver an additional signal to cells expressing FcγR. To demonstrate the utility of the vectors, the extracellular domain of human CD8α was expressed as a HCH2 polymer fusion protein. The fusion proteins were secreted in useful amounts from polyclonal cell lines established in Sf9 cells following transfection and selection with G418. The biological activity of the recombinant CD8α-HCH2 polymers was determined and compared to those of AIG and an anti-CD16 monoclonal antibody using an in vitro assay. The activity of the fusion proteins positively correlates to the number of HCH2 units. The largest polymer tested was severalfold more potent than AIG at similar concentrations. The HCH2 polymers described here represent a new strategy in the design of recombinant proteins for the therapeutic targeting of FcγR in autoimmune disorders.     

Rebmab200, a Humanized Monoclonal Antibody Targeting the Sodium Phosphate Transporter NaPi2b Displays Strong Immune Mediated Cytotoxicity against Cancer: A Novel Reagent for Targeted Antibody Therapy of Cancer

NaPi2b, a sodium-dependent phosphate transporter, is highly expressed in ovarian carcinomas and is recognized by the murine monoclonal antibody MX35. The antibody had shown excellent targeting to ovarian cancer in several early phase clinical trials but being murine the antibody''s full therapeutic potential could not be explored. To overcome this impediment we developed a humanized antibody version named Rebmab200, expressed in human PER.C6® cells and cloned by limiting dilution. In order to select a clone with high therapeutic potential clones were characterized using a series of physicochemical assays, flow cytometry, real-time surface plasmon resonance, glycosylation analyses, immunohistochemistry, antibody-dependent cell-mediated cytotoxicity, complement-dependent-cytotoxicity assays and quantitative PCR. Comparative analyses of Rebmab200 and MX35 monoclonal antibodies demonstrated that the two antibodies had similar specificity for NaPi2b by flow cytometry with a panel of 30 cell lines and maintained similar kinetic parameters. Robust and high producer cell clones potentially suitable for use in manufacturing were obtained. Rebmab200 antibodies were assessed by immunohistochemistry using a large panel of tissues including human carcinomas of ovarian, lung, kidney and breast origin. An assessment of its binding towards 33 normal human organs was performed as well. Rebmab200 showed selected strong reactivity with the tested tumor types but little or no reactivity with the normal tissues tested confirming its potential for targeted therapeutics strategies. The remarkable cytotoxicity shown by Rebmab200 in OVCAR-3 cells is a significant addition to the traits of stability and productivity displayed by the top clones of Rebmab200. Antibody-dependent cell-mediated toxicity functionality was confirmed in repeated assays using cancer cell lines derived from ovary, kidney and lung as targets. To explore use of this antibody in clinical trials, GMP production of Rebmab200 has been initiated. As the next step of development, Phase I clinical trials are now planned for translation of Rebmab200 into the clinic.     

Effects of the Deletion of Early Region 4 (E4) Open Reading Frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on Virus-Host Cell Interaction,Transgene Expression,and Immunogenicity of Replicating Adenovirus HIV Vaccine Vectors

The global health burden engendered by human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS) is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad)-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr) encoding full-length single-chain HIV_BaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC) we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1) through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and immunogenicity in a replicating Ad vector.