key: cord-0007374-dpoljl78
authors: Chiuchiolo, Maria J.; Boyer, Julie L.; Krause, Anja; Senina, Svetlana; Hackett, Neil R.; Crystal, Ronald G.
title: Protective Immunity against Respiratory Tract Challenge with Yersinia pestis in Mice Immunized with an Adenovirus-Based Vaccine Vector Expressing V Antigen
date: 2006-11-01
journal: J Infect Dis
DOI: 10.1086/507644
sha: b82a7e289836a97e6d2bc4edd7850bdbfa339160
doc_id: 7374
cord_uid: dpoljl78

The aerosol form of the bacterium Yersinia pestis causes the pneumonic plague, a rapidly fatal disease. At present, no plague vaccines are available for use in the United States. One candidate for the development of a subunit vaccine is the Y. pestis virulence (V) antigen, a protein that mediates the function of the Yersinia outer protein virulence factors and suppresses inflammatory responses in the host. On the basis of the knowledge that adenovirus (Ad) gene-transfer vectors act as adjuvants in eliciting host immunity against the transgene they carry, we tested the hypothesis that a single administration of a replication-defective Ad gene-transfer vector encoding the Y. pestis V antigen (AdsecV) could stimulate strong protective immune responses without a requirement for repeat administration. AdsecV elicited specific T cell responses and high IgG titers in serum within 2 weeks after a single intramuscular immunization. Importantly, the mice were protected from a lethal intranasal challenge of Y. pestis CO92 from 4 weeks up to 6 months after immunization with a single intramuscular dose of AdsecV. These observations suggest that an Ad gene-transfer vector expressing V antigen is a candidate for development of an effective anti-plague vaccine

At present, no plague vaccines are available in the United States. Several vaccines have been developed, including killed whole-cell formulations and the live attenuated EV76 vaccine [5] [6] [7] [8] . Although these vaccines have been used in humans, they offer low levels of protection, have numerous adverse side effects, and require frequent immunizations with consequent prolonged time to develop immunity [5] [6] [7] [8] . A promising subunit vaccine is based on the virulence (V) antigen (also referred to as "LcrV") [9] [10] [11] . V antigen is a 37-kDa multifunctional protein of Yersinia species encoded by the 70-kb low calcium response (lcr) plasmid. V antigen participates in the type III secretion system in Y. pestis regulating the production and facilitating the translocation of Yersinia outer proteins (Yops) with anti-host activity into the host cell [12, 13] . Active immunization with purifie V antigen or passive immunization with antiserum against V antigen provides protection against plague in mice [9, 10, 14, 15] . V antigen-based DNA vaccines are also being developed [16, 17] . These vaccines elicit low antibody titers, and protection against a Y. pestis challenge is reached only after several immunizations [18] .

The present study is focused on using replication-deficien adenovirus (Ad) gene-transfer vectors encoding V antigen to elicit protective immune responses against Y. pestis. Ad vectors are excellent candidates for vaccine platforms as they transfer genes effectively to antigen-presenting cells (APCs) in vivo, with consequent activation of APCs, thus conveying immune adjuvant properties and inducing strong, rapid humoral and cellular immune responses against the transgene product [19] [20] [21] [22] [23] [24] [25] [26] . On the basis of these considerations, we constructed an E1 Ϫ E3 Ϫ Ad-based vaccine vector that encodes a secreted human codonoptimized V antigen (AdsecV). The data demonstrate that AdsecV induces high IgG titers within 2 weeks after a single intramuscular immunization in mice. Importantly, mice immunized with a single intramuscular dose of AdsecV are protected from a lethal intranasal challenge of Y. pestis.

The Y. pestis V antigen gene (NCBI accession no. B33601) with mammalian-preferred codons was synthesized by overlap polymerase chain reaction and fused to the human Igk signal sequence for extracellular secretion. The V antigen gene was cloned into a recombinant Ad5-based vector (E1a, partial E1b, and partial E3 deletion), to generate AdsecV. AdNull was used as a control vector with identical backbone but no transgene [27] . The vectors were produced in 293 cells and were purifie by double CsCl gradient centrifugation [28] . Dosing was based on particle units (pu), the physical number of Ad particles as measured by spectrophotometry [29] .

Purificatio of recombinant V antigen. Recombinant V antigen was produced as a reagent for assessment of antibodies against V antigen. The V antigen gene was cloned into the pRSET expression plasmid (Invitrogen), and V antigen was purifie as a histidine-tag fusion by use of a Ni-NTA Superflo Column (Qiagen) under native conditions.

The A549 lung epithelial cell line (CCL185; American Type Culture Collection) was maintained in complete Dulbecco's modifie essential medium. Cells were infected with AdsecV or AdNull (500 pu/cell) in low-serum medium. Twenty-four hours after infection, cells and medium were collected, and proteins were separated by SDS-PAGE (Invitrogen) and transferred to a polyvinylidene fluorid membrane (BioRad Laboratories). For Western blot analysis, the membrane was probed with a 1:1000 dilution of a rabbit anti-V antigen antibody (provided by S. Bavari, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD). A peroxidase-conjugated anti-rabbit antibody (Santa Cruz Biotechnology) and a chemiluminescent peroxidase substrate (ECL+ reagent; Amersham Biosciences) were used for detection.

To assess V antigen expression by immunofluo escence, 24 h after infection, cells were fixe with 4% paraformaldehyde and blocked with 5% goat serum (Jackson ImmunoLabs), 1% bovine serum albumin in PBS with 0.05% saponin (Sigma-Aldrich), and 0.1% Triton X-100 (Sigma-Aldrich), followed by incubation with the rabbit anti-V antigen antibody diluted 1: 1000. A goat anti-rabbit secondary antibody conjugated to Alexa 488 fluo ophore (Jackson ImmunoLabs) was used at a fina concentration of 10 mg/mL. Nuclei were counterstained with 4 ,6-diamidino-2-phenylindole (Molecular Probes). The samples were observed by fluo escence microscopy with an Olympus IX70 inverted microscope (New York/New Jersey Scientific equipped with a ϫ60 PlanApo NA 1.4 objective, and digital image analysis was performed with Metamorph imaging software (version 4.6r9; Universal Imaging).

Mice. Female BALB/c mice were obtained from Taconic. Mice were housed under specific-pathogen-f ee conditions and were used at 7 weeks of age. Mice were immunized in a single vaccination by 2 intramuscular injections, with 50 mL of the vaccine preparation divided evenly between the quadriceps on each side. Ad vectors were diluted with saline to the specifie dose.

Transgene-specifi humoral responses. Serum samples from immunized mice were obtained from the tail vein, and anti-V antigen serum antibody titers were determined by ELISA. For vector dose response, serum samples were obtained from mice 4 weeks after immunization with AdsecV at doses ranging from 10 8 to 10 11 pu. For time-dependent anti-V antigen antibody titers, mice were immunized with a single administration of 10 9 pu of AdsecV, and serum samples were obtained before and at 1, 2, 4, 6, and 8 weeks after vaccination. For IgG subtypes, mice were vaccinated with 10 9 pu of AdsecV, and serum samples were obtained 5 weeks after immunization.

For ELISAs, microtiter plates (Corning) were coated with 0.5 mg of recombinant V antigen per well. After blocking, serum samples were added in sequential 2-fold dilutions starting at 1:20 and were incubated for 1 h at 23ЊC. An anti-mouse IgGhorseradish peroxidase conjugate (Sigma-Aldrich) was used at 1:10,000 dilution. Detection was accomplished using a peroxidase substrate (BioRad Laboratories). Absorbance at 415 nm was read using a microplate reader (BioRad Laboratories). Classspecifi anti-IgG antibodies (IgG1, IgG2a, IgG2b, and IgG3) were determined using the Mouse Typer isotyping panel (Bio-Rad Laboratories). Antibody titers were calculated on the basis of a log(optical density) Ϫ log(dilution) interpolation model and a cutoff value equal to 2-fold the absorbance of the background [30, 31] .

Transgene-specifi cellular responses. Mice were immunized intramuscularly ( ) with either saline, AdNull (10 11 n p 5 pu), or AdsecV (10 11 pu). The frequency of antigen-specifi T lymphocytes was determined using an interleukin (IL)-2-, in- terferon (IFN)-g-, and IL-4-specifi enzyme-linked immunospot (ELISPOT) assay (R&D Systems). Six days after administration of Ad, CD4 + or CD8 + T cells were purifie by negative depletion using SpinSep T cell subset purificatio kits (StemCell Technologies). Splenic dendritic cells (DCs) were purifie from naive mice by positive selection using CD11c MACS beads (Milentyi Biotec) and double purificatio over 2 MACS-LS columns (Milentyi Biotec). The purity of CD4 + T cells, CD8 + T cells, and DCs was assessed by staining with anti-CD4-phycoerythrin (PE), anti-CD8-PE, and anti-CD11c-PE antibodies (BD Biosciences), respectively. Cell purity evaluation and cell counts were performed using a FACScalibur flo cytometer running at a constant flo rate. For ELISPOT assays, 10 5 CD4 + or CD8 + T cells were incubated for 36 h with splenic DCs at a ratio of 4:1, with or without purifie V antigen. Spots were counted by computer-assisted ELISPOT image analysis (Zellnet Consulting).

Y. pestis CO92 challenge. The Y. pestis challenge studies were conducted at the Public Health Research Institute at the International Center for Public Health under biosafety level 3 conditions. Four weeks or 6 months after immunization, mice (10/group) were challenged intranasally with Y. pestis CO92. Y. pestis CO92 was grown aerobically in heart infusion broth (Difco) at 30ЊC and was diluted in saline solution at doses ranging from 10 3 to 10 6 cfu. Fifty microliters of bacterial suspension was used for intranasal infection of mice. Bacterial dose was controlled by plating on Yersinia selective agar (YSA; Oxoid). Survival was monitored daily for 15 days. From a subset of the mice that died after challenge, liver, spleen, and lungs were removed, homogenized in saline solution, and plated on YSA, to confi m that plague was the cause of death. A subset of the vaccinated mice that survived the challenge were killed 15 days after infection; liver, spleen, and lungs were removed, homogenized in saline solution, and plated on YSA to confi m that bacteria were not present in internal organs.

Statistical analyses. Data are presented as val-mean ‫ע‬ SE ues. For ELISPOT assays, statistical analyses were performed using 1-way analysis of variance followed by Fisher's protected least significan difference test. For survival comparison, Kaplan-Meier analysis was performed; reported P values are from Mantal-Cox analysis. Statistical significanc was determined at . P ! .05

AdsecV was analyzed in vitro by Western blot analysis. Twentyfour hours after infection of A549 cells with AdsecV, a protein with the expected size for V antigen (37 kDa) was identifie in medium and cell lysates by use of an anti-V antigen antibody (figu e 1A). The protein was not detected in cells infected with AdNull (the control vector) or in uninfected cells. The localization of V antigen in the AdsecV-infected cells was evaluated by indirect immunofluo escence. At 24 h, V antigen (figu e 1C) exhibits a broad, diffuse staining pattern as well as a bright, punctate, perinuclear staining pattern consistent with localization of V antigen in the endoplasmic reticulum and Golgi apparatus of the secretory pathway. The protein was not present in AdNull-infected cells (figu e 1B).

Humoral immune responses to AdsecV. To evaluate hu- To evaluate the kinetics of the anti-V antibody response, total IgG titers were measured at different time points after a single vaccination. After administration of a 10 9 -pu dose of AdsecV, anti-V antigen titers were detected in serum of immunized mice as early as 1 week (figu e 2B). The antibody titer reached a maximum level at 2 weeks and remained high through 8 weeks. Analysis of IgG subclasses at the 10 9 -pu AdsecV dose showed a strong response for both IgG1 and IgG2a and a lesser response for IgG2b and IgG3 (figu e 2C). Similar antibody titers and subtypes were observed with an Ad vector expressing a nonsecreted form of V antigen (data not shown). Anti-V antigen antibody after immunization with 10 9 pu of the nonsecreted form could be detected in serum at 2 weeks, with a titer of . Antibody levels re-mean ‫ע‬ SE 13,700 ‫ע‬ 2,900 mained high through week 8, as reported for AdsecV (figu e 2B). Because anti-V antigen antibodies in serum could already be detected 1 week after immunization with AdsecV and because titers were similar for both forms of the vaccine, we focused our study on the responses evoked by AdsecV.

Cellular immune responses to AdsecV. The frequency of T cell responses to V antigen in vaccinated mice was analyzed by ELISPOT assay. Six days after mice were immunized with 10 11 pu of AdsecV, purifie CD4 + and CD8 + T cells from the spleens of the vaccinated mice were stimulated with syngeneic DCs pulsed with V antigen, and cytokine production was assessed. V antigen-specifi IL-2 secretion ( , spots/ mean ‫ע‬ SE 52 ‫ע‬ 6 10 5 CD4 + T cells) (figu e 3A) as well as IFN-g secretion (mean ‫ע‬ SE, spots/10 5 CD4 + T cells) (figu e 3C) was 91 ‫ע‬ 3 significantl higher ( and , respectively) in P ! .005 P ! .0001 CD4 + T cells from the AdsecV-immunized mice than in the 2 control groups. In contrast, no significan differences were observed for V antigen-specifi IL-4 production (figu e 3B). CD8 + T cell activation was evaluated by V antigen-specifi IL-2 and IFN-g secretion. Both IL-2 ( , spots/10 5 CD8 + mean ‫ע‬ SE 19 ‫ע‬ 4 T cells) (figu e 4A) and IFN-g ( , spots/10 5 mean ‫ע‬ SE 24 ‫ע‬ 3 CD8 + T cells) (figu e 4B) responses were higher in the AdsecVvaccinated mice than in the 2 control groups ( ). The P ! .005 naive mice and the mice immunized with AdNull showed no significan signal of V antigen-induced cytokine production above background.

Protection against intranasal challenge with Y. pestis CO92. The ability of AdsecV to confer protective immunity was evaluated by challenging immunized mice with the fully virulent Y. pestis strain CO92. The mice received a single intramuscular administration of AdsecV at doses ranging from 10 8 to 10 11 pu. Four weeks after vaccination, the mice were infected intranasally with cfu of Y. pestis strain CO92. All (10/10) of 3 3 ϫ 10 the mice in the group vaccinated with 10 11 pu of AdsecV survived the Y. pestis challenge ( ) (figu e 5A). The survival P ! .005 of mice that were immunized with 10 10 pu of AdsecV ranged from 40% (4/10; ) (figu e 5A) to 60% (6/10) in a dif-P ! .05 ferent experiment (data not shown). The 10 9 -and 10 8 -pu doses were not protective; the mice in those groups died according to the same time frame as did the mice in the control groups that received either saline or 10 11 pu of AdNull. Assessment of the data at 15 days after challenge showed that the mortality of mice was dependent on the vaccine dose (figu e 5B).

To evaluate the protective capacity of the vaccine at different challenge doses, mice were infected intranasally with 10 3 -10 6 cfu of Y. pestis CO92 4 weeks after a single administration of 10 11 pu of AdsecV. Mice were protected at all doses (figu e 6). All AdsecV-immunized mice (10/10) survived the challenge with 10 3 ( ) and 10 4 ( ) cfu, whereas the naive P ! .005 P ! .0001 mice and the mice immunized with 10 11 pu of AdNull died within 3-5 days. At higher challenge doses, 80% (8/10) and 90% (9/10) of the mice survived after intranasal infection with 10 5 and 10 6 cfu Y. pestis CO92, respectively ( ). P ! .0001 The capacity of the vaccine to confer long-term protection was evaluated by challenge at 6 months after a single immunization with 10 11 pu of AdsecV. Anti-V antigen total IgG titers in immunized mouse serum before challenge were a mean ‫ע‬ of ( ). Mice were infected intra-SE 106,000 ‫ע‬ 16,500 n p 20 nasally with 10 4 or 10 6 cfu of Y. pestis CO92. All AdsecV-immunized mice (10/10) survived the challenge with 10 4 cfu and 90% (9/10) survived the challenge with 10 6 cfu ( , for P ! .0001 both doses) (figu e 7), whereas none of the 10 naive mice survived the challenge with 10 4 cfu Y. pestis CO92.

In the present study, AdsecV, a replication-defective Ad vector expressing a secreted form of the Y. pestis V antigen, was evaluated as a vaccine against plague. The V antigen sequence in AdsecV was targeted for extracellular expression by an Igk Figure 6 . Yersinia pestis CO92 challenge dose response of AdsecV-immunized mice. Mice were immunized intramuscularly with either saline (naive mice), the control vector AdNull (10 11 particle units [pu]), or AdsecV (10 11 pu) and were challenged 4 weeks later by intranasal administration of 10 3 -10 6 cfu of Y. pestis CO92. Data are from 1 experiment (n p 10 mice/group). Survival at 15 days is plotted against dose. Because all naive and AdNull-immunized mice died at a dose of 10 4 cfu, higher doses of Y. pestis were not assessed in the 2 control groups. For the mice that received 10 3 cfu and for the mice that received the higher challenge doses, P ! and , respectively, compared with the 2 control groups. .005 P ! .0001 Figure 7 . Long-term protection of AdsecV-immunized mice. Mice were immunized intramuscularly with either saline (naive mice) or AdsecV (10 11 particle units [pu]) and were challenged 6 months later by intranasal administration of 10 4 or 10 6 cfu of Yersinia pestis CO92 ( , com-P ! .0001 pared with the naive mice). Survival is plotted as a function of time. Data are from 1 experiment ( mice/group). n p 10 secretion signal, and V antigen expression in infected cells was confi med by Western blot analysis and indirect immunoflu orescence. After a single intramuscular immunization with AdsecV, mice developed strong humoral responses within 2 weeks, with anti-V antigen IgG titers predominantly of the IgG2a and IgG1 subtypes, suggesting a strong Th1 and Th2 response. The cellular immune responses observed in splenic T cells from vaccinated mice were V antigen-specifi Th1 helper (CD4 + ) and CD8 + responses. Most importantly, immunized mice were protected from an intranasal challenge with a lethal dose of 10 6 cfu of Y. pestis CO92, from 4 weeks through 6 months after a single administration of the vaccine.

Y. pestis vaccines. Plague is one of the most devastating acute infectious diseases experienced by humankind [1] [2] [3] [4] [5] . Antibiotics are only marginally effective once symptoms of pneumonic plague develop; moreover, some antibiotic-resistant isolates have been identified Given these characteristics, there is concern that an aerosolized form of Y. pestis may be exploited as a bioweapon.

There is no licensed Y. pestis vaccine for use in the United States. Killed whole-cell vaccines have been used since the late 1890s [5] [6] [7] [8] . Although these vaccines have been shown to protect against the bubonic form of the disease, they do not protect against pneumonic plague. These vaccines also have disadvantages, such as a significan incidence of transient local and systemic adverse side effects and the need for frequent boosting to maintain adequate immunity [5, 7, 8] . A live attenuated vaccine based on the pigmentation-negative Y. pestis strain EV76 has been available since 1908 [6] . This vaccine had questionable efficac in evoking effective immune responses in humans and presents the risk for reversion to virulence in vivo.

In recent years, the development of a safe and effective plague vaccine has been focused on using recombinant protein subunits of Y. pestis [5, [9] [10] [11] . Several virulence factors have been identifie as possible vaccine candidates, but the most promising are the V antigen and the F1 protein [7] [8] [9] [10] [11] [32] [33] [34] . Antibodies against V and F1 confer protection against both bubonic and pneumonic plague in mice, guinea pigs, and nonhuman primates [9-11, 14, 15, 33, 35] . Recent studies have shown that a single intramuscular immunization with both recombinant antigens delivered with adjuvants and combined in a molar ratio of 2:1 (F1:V antigen) protected mice against an aerosol challenge with Y. pestis [10, 11, [36] [37] [38] [39] . Although this protection was correlated with high IgG levels [40] , it has been suggested that nonhumoral immune responses also participate, because immunized IL-4-deficien mice, which do not mount effective humoral immune responses, have been shown to be protected against plague [41] . Also, immunization with the combined subunits failed to protect signal transducer and activator of transcription (Stat) 4 Ϫ/Ϫ mice against plague; Stat 4 Ϫ/Ϫ mice are diminished in their capacity to mount type 1 cytokine responses [42] . It was shown recently that cellular immunity in the absence of antibody can protect against pulmonary Y. pestis infection; transfer of Y. pestis-primed T cells to naive B cell-deficien mMT mice protected the mice against a Y. pestis challenge [43] .

V antigen-based vaccines. V antigen is a good candidate for Y. pestis vaccine development because it can protect from infection with either F1 + or F1 Ϫ strains [44] . V antigen plays important roles in the virulence of plague. It participates in the regulation and translocation of the effector proteins Yops into host cells through a type III secretion system. Yops virulence factors produce cytoskeletal rearrangements and apoptosis in macrophage-like cells, allowing bacteria to escape phagocytosis and to proliferate extracellularly [12, 13, 45, 46] . In addition, purifie V antigen has been shown to suppress the normal inflammato y response in the host by down-regulating the expression of tumor necrosis factor (TNF)-a and IFN-g, which promotes the production of IL-10 by macrophages, and by inhibiting the chemotaxis of neutrophils [47] [48] [49] [50] .

The basis for the protection conferred by the recombinant V antigen vaccine is not well known. The correlation with high IgG titers and protection of immunodeficien SCID/Beige mice against pneumonic plague by passive transfer of anti-V antigen-specifi immune serum [14, 15, 51] suggests that anti-V antigen antibodies play a significan role. It has been shown that anti-V antigen antibody administered during infections of mice with Y. pestis restore the production of TNF-a and IFNg [49] , and in vitro assays have shown that anti-V antigen antibodies can partially block the delivery of Yops (and the consequent downstream effects of Yops) in infected macrophage-like cells [52] . Thus, the protection conferred by a V antigen vaccine might be achieved by opsonization through antibody association with surface V antigen on Y. pestis, by blocking the delivery of Yops to host cells, by preventing early bacterial growth in macrophages, and/or by neutralizing the immunomodulatory activity of V antigen, allowing the host to mount an inflammato y response.

Ad vectors as vaccine platforms. Recombinant Ad vectors are attractive for vaccine strategies against pathogens for many reasons. They are stable, easy to manipulate, can be produced inexpensively at high titer, and can be purifie by commonly available methods [53] . Ad vectors are capable of delivering genes to a broad variety of cell types, and relevant to their use as a vaccine is their ability to infect DCs and other APCs in vivo. The Ad vector itself may act as adjuvant by inducing a strong inflammato y response at the injection site and by promoting the differentiation of immature DCs into professional APCs [54] [55] [56] . Recombinant Ad vectors expressing a wide variety of pathogen-specifi genes have been used in vaccination studies in rodents, canines, and nonhuman primates [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] . Ad vectors induce protective adaptive immune responses against the transgene product very rapidly after a single application [19] [20] [21] [22] [23] [24] [25] [26] . This feature is particularly useful for postexposure vaccination or to combat infectious agents that cause infrequent but rapidly spreading outbreaks associated with high mortality.

The present evaluation of the efficac of an Ad vaccine, AdsecV, expressing the Y. pestis V antigen demonstrated the induction of rapid protective humoral and cellular immune responses. AdsecV antibody responses were elicited rapidly (within 2 weeks after administration) and showed the characteristic Th1/Th2 responses elicited by Ad vectors. Most importantly, the immune responses evoked by AdsecV were sufficien to protect immunized mice against an intranasal challenge with a fully virulent strain of Y. pestis. Together, these data suggest that AdsecV is a promising vaccine candidate for protection against plague.

Emergency preparedness and response: bioterrorism agents/disease

Yersinia pestis-etiologic agent of plague

Plague as a biological weapon: medical and public health management. Working Group on Civilian Biodefense

Yersiniosis I: microbiological and clinicoepidemiological aspects of plague and non-plague Yersinia infections

Vaccines. Philadelphia: WB Saunders

A comparison of plague vaccine, USP and EV76 vaccine induced protection against Yersinia pestis in a murine model

Vaccination against bubonic and pneumonic plague

Yersinia pestis (plague) vaccines

Recombinant V antigen protects mice against pneumonic and bubonic plague caused by F1-capsule-positive and -negative strains of Yersinia pestis

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A new improved sub-unit vaccine for plague: the basis of protection

The V-antigen of Yersinia is surface exposed before target cell contact and involved in virulence protein translocation

The Yersinia Yop virulon: LcrV is required for extrusion of the translocators YopB and YopD

The SCID/Beige mouse as a model to investigate protection against Yersinia pestis

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A DNA vaccine producing LcrV antigen in oligomers is effective in protecting mice from lethal mucosal challenge of plague

Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors

Adenoviruses as gene-delivery vehicles

Cytotoxic T lymphocyte responses to proteins encoded by heterologous transgenes transferred in vivo by adenoviral vectors

Antivector and antitransgene host responses in gene therapy

Adenovirus hexon protein is a potent adjuvant for activation of a cellular immune response

Immune responses to gene therapy vectors: influenc on vector function and effector mechanisms

Adenovirus vector-based vaccines for human immunodeficienc virus type 1

Adenovirus-based genetic vaccines for biodefense

Modulation of gene expression after replication-deficient recombinant adenovirus-mediated gene transfer by the product of a second adenovirus vector

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Comparisons of standard curve-fittin methods to quantitate Neisseria meningitidis group A polysaccharide antibody levels by enzyme-linked immunosorbent assay

Protection against anthrax lethal toxin challenge by genetic immunization with a plasmid encoding the lethal factor protein

Protective efficac of recombinant Yersinia outer proteins against bubonic plague caused by encapsulated and nonencapsulated Yersinia pestis

Protective efficac of a fully recombinant plague vaccine in the guinea pig

Yersinia outer proteins (YOPS)

are antigenic but non-protective compared to V antigen, in a murine model of bubonic plague

Public workshop on animals models and correlates of protection for plague vaccines

Shortand long-term efficac of single-dose subunit vaccines against Yersinia pestis in mice

Fraction 1 capsular antigen (F1) purificatio from Yersinia pestis CO92 and from an Escherichia coli recombinant strain and efficac against lethal plague challenge

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An IgG1 titre to the F1 and V antigens correlates with protection against plague in the mouse model

The F1 and V subunit vaccine protects against plague in the absence of IL-4 driven immune responses

Stat 4 but not Stat 6 mediated immune mechanisms are essential in protection against plague

Cell-mediated protection against pulmonary Yersinia pestis infection

Relationship between virulence and immunity as revealed in recent studies of the F1 capsule of Yersinia pestis

The virulence plasmid of Yersinia, an antihost genome

The V antigen of Yersinia pestis regulates Yop vectorial targeting as well as Yop secretion through effects on YopB and LcrG

Association between virulence of Yersinia pestis and suppression of gamma interferon and tumor necrosis factor alpha

V antigen of Yersinia pestis inhibits neutrophil chemotaxis

Suppression of cytokines in mice by protein A-V antigen fusion peptide and restoration of synthesis by active immunization

Yersinia enterocolitica evasion of the host innate immune response by V antigen-induced IL-10 production of macrophages is abrogated in IL-10-deficien mice

Synergistic protection of mice against plague with monoclonal antibodies specifi for the F1 and V antigens of Yersinia pestis

Antibody against V antigen prevents Yop-dependent growth of Yersinia pestis

Adenovirus vectors for gene therapy

Recombinant adenovirus Adenovirus-Based Y. pestis Vaccine • JID 2006:194 (1 November) • 1257 induces maturation of dendritic cells via an NF-kappaB-dependent pathway

Modificatio to the capsid of the adenovirus vector that enhances dendritic cell infection and transgenespecifi cellular immune responses

Acute cytokine response to systemic adenoviral vectors in mice is mediated by dendritic cells and macrophages

Adenoviruses as vaccine vectors

Neutralizing antibodies elicited by immunization of monkeys with DNA plasmids and recombinant adenoviral vectors expressing human immunodeficienc virus type 1 proteins

A simian replicationdefective adenoviral recombinant vaccine to HIV-1 gag

Immunogenicity of recombinant human adenovirus-human immunodeficienc virus vaccines in chimpanzees

Immunogenicity of recombinant adenovirus-human immunodeficienc virus vaccines in chimpanzees following intranasal administration

Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-viru immunity

Immunogenicity and efficac testing in chimpanzees of an oral hepatitis B vaccine based on live recombinant adenovirus

Enhanced induction of hepatitis C virus-specifi cytotoxic T lymphocytes and protective efficac in mice by DNA vaccination followed by adenovirus boosting in combination with the interleukin-12 expression plasmid

Immunogenicity of an E1-deleted recombinant human adenovirus against rabies by different routes of administration

A replication-defective human adenovirus recombinant serves as a highly efficaciou vaccine carrier

Development of a preventive vaccine for Ebola virus infection in primates

Accelerated vaccination for Ebola virus haemorrhagic fever in non-human primates

Effects of a SARS-associated coronavirus vaccine in monkeys

Replication-defective adenoviral vaccine vector for the induction of immune responses to dengue virus type 2

Enhanced protective immunity against malaria by vaccination with a recombinant adenovirus encoding the circumsporozoite protein of Plasmodium lacking the GPI-anchoring motif

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We thank T. Niven, for performing the antibody assays; Dr. D. Perlin and the staff of the Public Health Research Institute at the International Center for Public Health, for help with the biosafety level 3 studies; and N. Mohamed, for help in preparing the manuscript.