Ju Joh

Epitopes expressed in different adenovirus capsid proteins induce different levels of epitope-specific immunity.

ABSTRACT On the basis of the concept that the capsid proteins of adenovirus (Ad) gene transfer vectors can be genetically manipulated to enhance the immunogenicity of Ad-based vaccines, the present study compared the antiantigen immunogenicity of Ad vectors with a common epitope of the hemagglutinin (HA) protein of the influenza A virus incorporated into the outer Ad capsid protein hexon, penton base, fiber knob, or protein IX. Incorporation of the same epitope into the different capsid proteins provided insights into the correlation between epitope position and antiepitope immunity. Following immunization of three different strains of mice (C57BL/6, BALB/c, and CBA) with either an equal number of Ad particles (resulting in a different total HA copy number) or different Ad particle numbers (to achieve the same HA copy number), the highest primary (immunoglobulin M [IgM]) and secondary (IgG) anti-HA humoral and cellular CD4 gamma interferon and interleukin-4 responses against HA were always achieved with the Ad vector carrying the HA epitope in fiber knob. These observations suggest that the immune response against an epitope inserted into Ad capsid proteins is not necessarily dependent on the capsid protein number and imply that the choice of incorporation site in Ad capsid proteins in their use as vaccines needs to be compared in vivo.

Protective Immunity to Pseudomonas aeruginosa Induced with a Capsid-Modified Adenovirus Expressing P. aeruginosa OprF

ABSTRACT This study focuses on the development of a new clinical vaccine candidate (AdOprF.RGD.Epi8) against Pseudomonas aeruginosa using an E1− E3− adenovirus (Ad) vector expressing OprF (AdOprF.RGD.Epi8) and modifications of the Ad genome providing two capsid changes: (i) modification of the Ad hexon gene to incorporate an immune-dominant OprF epitope (Epi8) into loop 1 of the hexon, enabling repeat administration to boost the anti-OprF immune response, and (ii) modification of the fiber gene to incorporate an integrin-binding RGD sequence to enhance gene delivery to antigen-presenting cells. Western analysis confirmed that AdOprF.RGD.Epi8 expresses OprF, contains Epi8 in the hexon protein, and enhances gene transfer to dendritic cells compared to AdOprF, a comparable Ad vector expressing OprF with an unmodified capsid. Intramuscular immunization of C57BL/6 mice with AdOprF.RGD.Epi8 resulted in the generation of anti-OprF antibodies at comparable levels to those induced following immunization with AdOprF, but immunization with AdOprF.RGD.Epi8 was associated with increased CD4 and CD8 gamma interferon T-cell responses against OprF as well as increased survival against lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeat administration of AdOprF.RGD.Epi8 resulted in boosting of the humoral anti-OprF response as well as increased protection, whereas no boosting could be achieved with repeat administration of AdOprF. This suggests that the capsid-modified AdOprF.RGD.Epi8 vector is a more effective immunogen compared to a comparable wild-type Ad capsid, making it a good candidate for an anti-P. aeruginosa vaccine.

Genetic delivery of an anti-RSV antibody to protect against pulmonary infection with RSV

Respiratory syncytial virus (RSV) is a common cause of severe lower respiratory tract infections. Protection against infection with RSV can be achieved by monthly administration of the humanized monoclonal antibody palivizumab. The present study analyzes if genetic delivery of a murine version of palivizumab by single administration would achieve high-level and sustained antibody expression to protect mice against pulmonary infection with RSV. A murine version of the palivizumab antibody was constructed by replacing the human sequences with sequences from the constant region of a murine IgG1 antibody, while preserving the complementarity-determining region. As a proof-of-principle to test the validity of the strategy, the coding sequence for the heavy and light chains were cloned into a replication-defective serotype 5 human adenovirus vector (AdalphaRSV). Antibody expression and specificity for RSV was confirmed by Western analysis. To determine if AdalphaRSV would mediate production of anti-RSV antibodies in vivo, 5x10(10) particle units of AdalphaRSV or a control vector without transgene (AdNull), were administered intravenously to BALB/c mice. RSV neutralizing antibodies were detected in the serum after 4 days in mice receiving AdalphaRSV but not in AdNull-infected or naive mice (p<0.05). The mice that had received AdalphaRSV had at least 5.4-fold lower RSV titers in the lung 4 days following intranasal challenge with RSV compared to the AdNull or naive group (p<0.01). To evaluate long-term protection, the antibody construct was expressed in a non-human primate serotype rh.10 adeno-associated virus vector (AAVrh.10alphaRSV). RSV neutralizing antibodies were detected in serum and bronchoalveolar lavage fluid for up to 21 wk following intrapleural administration of AAVrh.10alphaRSV, but not with a control AAV vector expressing an unrelated transgene (AAVrh.10alpha1AT). Following challenge with RSV at 7 or 21 wk, 14.3-fold and 10.6-fold lower RSV titers were observed after 4 days in the lungs of mice that had received AAVrh.10alphaRSV compared to AAVrh.10alpha1AT (p<0.05). Together these data demonstrate that a gene transfer strategy for delivery of an anti-RSV antibody can generate protective immunity in mice against RSV infection in the respiratory tract and may provide an alternative to the administration of the antibody itself.

Protective anti-Pseudomonas aeruginosa humoral and cellular mucosal immunity by AdC7-mediated expression of the P. aeruginosa protein OprF

Replication-deficient adenoviral (Ad) vectors are an attractive platform for a vaccine against lung infections caused by Pseudomonas aeruginosa. Ad vectors based on non-human serotypes have been developed to circumvent the problem of pre-existing anti-Ad immunity in humans. The present study analyzes the anti-P. aeruginosa systemic and lung mucosal immunity elicited by a non-human primate-based AdC7 vector expressing the outer membrane protein F (AdC7OprF) of P. aeruginosa. Intramuscular immunization of mice with AdC7OprF induced similar levels of serum and mucosal anti-OprF IgG and increased levels of anti-OprF IgA in lung epithelial lining fluid (ELF) compared to immunization with a human serotype Ad5OprF vector (p>0.05). OprF-specific INF-γ in splenic T cells stimulated with OprF-pulsed syngeneic splenic dendritic cells (DC) was similar following immunization with AdC7OprF compared to Ad5OprF (p>0.05). In contrast, OprF-specific INF-γ responses in lung T cells stimulated with either spleen or lung DC were increased following immunization with AdC7OprF compared to Ad5OprF (p<0.05). Interestingly, direct administration of AdC7OprF to the respiratory tract resulted in an increase of OprF-specific IgG in serum, OprF-specific IgG and IgA in lung ELF, and OprF-specific INF-γ in lung T-cells compared to immunization with Ad5OprF, and survival following challenge with a lethal dose of P. aeruginosa. These data demonstrate that systemic or lung mucosal immunization with an AdC7-based vaccine vector induces superior pulmonary humoral and cellular anti-transgene immunity compared to immunization with an Ad5-based vector and favors AdC7-based vectors as vaccines to induce lung mucosal immunity.

Expression of B-Cell Activating Factor Enhances Protective Immunity of a Vaccine against Pseudomonas aeruginosa

ABSTRACT B-cell activating factor (BAFF), a member of the TNF family, is a potent cytokine with stimulatory effects on B and T cells. To evaluate the potential of transient overexpression of BAFF to enhance vaccine immunogenicity, a replication-deficient adenovirus expressing full-length murine BAFF (AdBAFF) was tested in a mouse vaccine model against Pseudomonas aeruginosa. When coadministered with heat-killed P. aeruginosa, AdBAFF mediated a significant increase in anti-P. aeruginosa-specific serum and lung mucosal antibodies and resulted in improved protection against a lethal respiratory challenge with P. aeruginosa. This effect was independent of the site of administration of AdBAFF and was observed both when AdBAFF was given simultaneously with heat-killed P. aeruginosa as well as when AdBAFF was administered 4 weeks after immunization with heat-killed P. aeruginosa. These data demonstrate that a temporal increase in systemic BAFF levels is able to augment a P. aeruginosa-specific immune response upon immunization with heat-killed P. aeruginosa, suggesting that the immune-stimulatory effects of BAFF may be exploited as a molecular adjuvant for genetic vaccines.

Absence of vaccine-enhanced RSV disease and changes in pulmonary dendritic cells with adenovirus-based RSV vaccine

The development of a vaccine against respiratory syncytial virus (RSV) has been hampered by the risk for vaccine-enhanced RSV pulmonary disease induced by immunization with formalin-inactivated RSV (FIRSV). This study focuses on the evaluation of vaccine-enhanced pulmonary disease following immunization with AdF.RGD, an integrin-targeted adenovirus vector that expresses the RSV F protein and includes an RGD (Arg-Gly-Asp) motif. Immunization of BALB/c mice with AdF.RGD, resulted in anti-RSV protective immunity and induced increased RSV-specific IFN-γ T cell responses compared to FIRSV. RSV infection 5 wk after immunization with FIRSV induced pulmonary inflammatory responses in the lung, that was not observed with AdF.RGD. Additionally, In the FIRSV-immunized mice following infection with RSV, pulmonary DC increased and Tregs decreased. This suggests that distinct responses of pulmonary DC and Tregs are a features of vaccine-enhanced RSV disease and that immunization with an RGD-modified Ad vaccine does not trigger vaccine-enhanced disease.

Overexpression of Sonic Hedgehog in the Lung Mimics the Effect of Lung Injury and Compensatory Lung Growth on Pulmonary Sca-1 and CD34 Positive Cells

Cells localized in the bronchioalveolar duct junction of the murine lung have been identified as potential bronchioalveolar stem cells. Based on the surface marker expression, two main phenotypes have been proposed: Sca-1(+), CD34(+), CD45(-), Pecam(-) and Sca-1(low), CD34(-) CD45(-), Pecam(-) cells. An increase in the number of Sca-1(+), CD34(+) CD45(-), Pecam(-) cells and activation of the sonic hedgehog (Shh) pathway was observed following unilateral pneumonectomy and naphthalene-induced airway injury. Overexpression of Shh in the respiratory tract also resulted in an increase of this cell population. Syngeneic transplantation of beta-galactosidase-expressing bone marrow cells demonstrated that the increase of Sca-1(+), CD34(+), CD45(-), Pecam(-) cells in the lung was a result of local proliferation. Intratracheal administration of purified Shh-stimulated Sca-1(+), CD45(-), Pecam(-) cells coexpressing CD34 to syngeneic mice following pneumonectomy resulted in engraftment of these cells predominantly in the airways for up to 3 months, whereas Sca-1(-), CD45(-), Pecam(-) cells did not engraft. This study suggests that local Sca-1(+), CD34(+), CD45(-), Pecam(-) cells are stimulated during compensatory lung growth, following airway injury and overexpression of Shh and have some potential to engraft in the airways, without showing clonal properties in vivo.