and SC-Ad
Single-Cycle Replicating Adenovirus Vectors As Mucosal Vaccine Platforms Stephanie Anguiano-Zarate1, Catherine M. Crosby2, William Matchett, and Michael A. Barry3 Clinical Translational Sciences Graduate Program1, Virology and Gene Therapy Graduate Program2, Division of Infectious Diseases, Department of Medicine, Department of Immunology, and Department of Molecular Medicine3
Many viral and non-viral vectors are being proposed as potential vaccines. Adenoviral (Ad) vectors are arguably one of the most potent genebased vaccine vectors available today.
SC-Ads retain their E1 gene allowing them to undergo full DNA replication and transgene amplification after cell infection. However, they are deleted for the late gene IIIA for a virion cement protein. IIIA deletion blocks their ability to assemble functional infectious Ad virions. SCAds amplify genes and immune responses, but avoid the risk of adenovirus infections.
Replication-Competent Ads (RC-Ads, Fig. 1). In contrast, replication-competent Ads (RC-Ads) retain their E1 gene and can replicate their DNA and the antigen genes they carry 10,000-fold in an infected cell. RC-Ads can produce 100's of times the amount of antigen and drive substantially stronger immune responses than E1-deleted vectors (9-21). While RC-Ad are potent, they risk causing frank adenovirus infections in vaccinees or healthcare workers.
Fig. 2. Comparison of GFP Transduction by RDand SC-Ad6. GFP by fluorescence microscopy after infection of primary human lung cells with the indicated viral particles per cell of GFPLuc viruses. Single Intranasal Immunization with RD- and SC-Ad in Hamsters For example, after single intranasal immunization, only SC-Ad generated antibodies in vaginal washes that rose over 6 months (RD and RC did not) (Fig. 3C).
Fig. 1. Representation of Different Ad Vectors. RC- and SC-Ad produce more GFP in primary human cells with 33-fold fewer virions than RDAd and 30 to 300-fold higher luciferase than RDAd6 (Fig. 2, 3A, and B). From a vector production standpoint, a 33-fold improvement in antigen expression per unit virus by SC-Ad could translate into getting 33 times as many doses out of an SC-Ad GMP production than for an RD-Ad production. SC-Ads generate more robust and more persistent immune responses than either RD-Ad or RC-Ads after single immunization. (Fig. 3)
Single Intranasal Immunization with SC-Ads Expressing Influenza HA or Ebola Glycoprotein. SC-Ad carrying influenza hemagglutinin (HA) generated markedly higher expression of HA than RD-Ad (Fig. 3E). This higher expression by SC-Ad produced significantly higher anti-influenza binding antibodies and hemagglutination inhibition (HAI) antibodies than RD-Ad in hamsters at doses as low as 108 virus particles (vp) (Fig. 3D and not shown). SC-Ads expressing HA and SC-Ad expressing the Ebola Zaire KJ660346 glycoprotein were next used as controls for each other (Fig. 4). A single 109 vp intranasal immunization with both produced antibodies that rose and persisted for 6 months.
3
SC-Ad6-EBOV gp SC-Ad6-Influenza HA PBS
2
1
0
Comparison of RD- and SC-Ad Sublingual Vaccines in Rhesus Macaques. When rhesus macaques were immunized sublingually a single time with 1x1011 vp of RD-Ad6 and SC-Ad6, SCAd induced higher GFP antibodies than RD-Ad6 that climbed over 9 weeks (Fig. 3E).
However, in most cases, these Ad vaccines are actually not the most potent Ad vector platform. Replication-defective Ads (RD-Ads, Fig. 1). Most tested Ad vaccines are replication-defective Ads (RD-Ads). These are defective by deletion of their E1 gene to prevent the ability of the vaccine to cause an adenovirus infection in the recipient. An E1-deleted Ad vaccine infects a cell, delivers a single copy of an antigen gene, and expresses "1X" protein.
Anti-HA Antibodies (OD450)
Most infections start at mucosal surfaces. Combating the earliest events in pathogen entry is one strategy to combat infectious diseases, since neutralizing the few pathogens present at the beginning of an infection may be easier than when they spread to the rest of the body. Vaccines that also stimulate the systemic immune system are likely also needed to control pathogen spread once the mucosal barrier is breached.
Anti-EBOV Antibodies (OD450)
Introduction
New Single-cycle Ads (SC-Ads, Fig. 1). To harness antigen gene replication and amplify immune responses while avoiding the risk of adenovirus infections, the Barry lab developed single-cycle adenoviruses (SC-Ads).
Fig. 3. Comparison of SC-Ad with RD and RC-Ads. A) Luciferase after infection of the indicated cells with GFPLuc viruses (human=A549, macaque=FrhK4, hamster=HaK, mouse=Hepa1-6). B) Luciferase imaging Ad6after intranasal immunization in mice and hamsters. C) ELISA of vaginal wash anti-GFP antibodies after single intranasal administration (n=5). D) Anti-GFP serum antibodies in macaques immunized a single time with RD- and SC-Ad (n=3). E) Western of influenza HA 24 h after infection of A549 cells. F) Serum influenza hemagglutination inhibition titers after single intranasal immunization in hamsters
0 4 8 12 16 20 24 28 32 Time After Single Intranasal Immunization (Weeks) 0.8 SC-Ad6-EBOV gp SC-Ad6-Influenza HA
0.6 0.4 0.2 0.0
0 4 8 12 16 20 24 28 32 Time After Single Intranasal Immunization (Weeks)
Fig. 5. Single Immunization with SC-Ads vs. Influenza or Ebola. Groups of 5 hamsters were immunized intranasally a single time with 109 vp of SC-Ad6-HA or EBOVgp and sera were used for ELISAs.
Conclusions • SC-Ad vectors amplify antigen genes to amplify vaccine responses.
• SC-Ads are more potent than current RD-Ads. • SC-Ads generate long-lasting systemic or mucosal antibody responses after single mucosal immunization in mice, hamsters, and macaques
• SC-Ads may be useful mucosal vaccines.
Many viral and non-viral vectors are being proposed as potential vaccines. Adenoviral (Ad) vectors are arguably one of the most potent genebased vaccine vectors available today.
SC-Ads retain their E1 gene allowing them to undergo full DNA replication and transgene amplification after cell infection. However, they are deleted for the late gene IIIA for a virion cement protein. IIIA deletion blocks their ability to assemble functional infectious Ad virions. SCAds amplify genes and immune responses, but avoid the risk of adenovirus infections.
Replication-Competent Ads (RC-Ads, Fig. 1). In contrast, replication-competent Ads (RC-Ads) retain their E1 gene and can replicate their DNA and the antigen genes they carry 10,000-fold in an infected cell. RC-Ads can produce 100's of times the amount of antigen and drive substantially stronger immune responses than E1-deleted vectors (9-21). While RC-Ad are potent, they risk causing frank adenovirus infections in vaccinees or healthcare workers.
Fig. 2. Comparison of GFP Transduction by RDand SC-Ad6. GFP by fluorescence microscopy after infection of primary human lung cells with the indicated viral particles per cell of GFPLuc viruses. Single Intranasal Immunization with RD- and SC-Ad in Hamsters For example, after single intranasal immunization, only SC-Ad generated antibodies in vaginal washes that rose over 6 months (RD and RC did not) (Fig. 3C).
Fig. 1. Representation of Different Ad Vectors. RC- and SC-Ad produce more GFP in primary human cells with 33-fold fewer virions than RDAd and 30 to 300-fold higher luciferase than RDAd6 (Fig. 2, 3A, and B). From a vector production standpoint, a 33-fold improvement in antigen expression per unit virus by SC-Ad could translate into getting 33 times as many doses out of an SC-Ad GMP production than for an RD-Ad production. SC-Ads generate more robust and more persistent immune responses than either RD-Ad or RC-Ads after single immunization. (Fig. 3)
Single Intranasal Immunization with SC-Ads Expressing Influenza HA or Ebola Glycoprotein. SC-Ad carrying influenza hemagglutinin (HA) generated markedly higher expression of HA than RD-Ad (Fig. 3E). This higher expression by SC-Ad produced significantly higher anti-influenza binding antibodies and hemagglutination inhibition (HAI) antibodies than RD-Ad in hamsters at doses as low as 108 virus particles (vp) (Fig. 3D and not shown). SC-Ads expressing HA and SC-Ad expressing the Ebola Zaire KJ660346 glycoprotein were next used as controls for each other (Fig. 4). A single 109 vp intranasal immunization with both produced antibodies that rose and persisted for 6 months.
3
SC-Ad6-EBOV gp SC-Ad6-Influenza HA PBS
2
1
0
Comparison of RD- and SC-Ad Sublingual Vaccines in Rhesus Macaques. When rhesus macaques were immunized sublingually a single time with 1x1011 vp of RD-Ad6 and SC-Ad6, SCAd induced higher GFP antibodies than RD-Ad6 that climbed over 9 weeks (Fig. 3E).
However, in most cases, these Ad vaccines are actually not the most potent Ad vector platform. Replication-defective Ads (RD-Ads, Fig. 1). Most tested Ad vaccines are replication-defective Ads (RD-Ads). These are defective by deletion of their E1 gene to prevent the ability of the vaccine to cause an adenovirus infection in the recipient. An E1-deleted Ad vaccine infects a cell, delivers a single copy of an antigen gene, and expresses "1X" protein.
Anti-HA Antibodies (OD450)
Most infections start at mucosal surfaces. Combating the earliest events in pathogen entry is one strategy to combat infectious diseases, since neutralizing the few pathogens present at the beginning of an infection may be easier than when they spread to the rest of the body. Vaccines that also stimulate the systemic immune system are likely also needed to control pathogen spread once the mucosal barrier is breached.
Anti-EBOV Antibodies (OD450)
Introduction
New Single-cycle Ads (SC-Ads, Fig. 1). To harness antigen gene replication and amplify immune responses while avoiding the risk of adenovirus infections, the Barry lab developed single-cycle adenoviruses (SC-Ads).
Fig. 3. Comparison of SC-Ad with RD and RC-Ads. A) Luciferase after infection of the indicated cells with GFPLuc viruses (human=A549, macaque=FrhK4, hamster=HaK, mouse=Hepa1-6). B) Luciferase imaging Ad6after intranasal immunization in mice and hamsters. C) ELISA of vaginal wash anti-GFP antibodies after single intranasal administration (n=5). D) Anti-GFP serum antibodies in macaques immunized a single time with RD- and SC-Ad (n=3). E) Western of influenza HA 24 h after infection of A549 cells. F) Serum influenza hemagglutination inhibition titers after single intranasal immunization in hamsters
0 4 8 12 16 20 24 28 32 Time After Single Intranasal Immunization (Weeks) 0.8 SC-Ad6-EBOV gp SC-Ad6-Influenza HA
0.6 0.4 0.2 0.0
0 4 8 12 16 20 24 28 32 Time After Single Intranasal Immunization (Weeks)
Fig. 5. Single Immunization with SC-Ads vs. Influenza or Ebola. Groups of 5 hamsters were immunized intranasally a single time with 109 vp of SC-Ad6-HA or EBOVgp and sera were used for ELISAs.
Conclusions • SC-Ad vectors amplify antigen genes to amplify vaccine responses.
• SC-Ads are more potent than current RD-Ads. • SC-Ads generate long-lasting systemic or mucosal antibody responses after single mucosal immunization in mice, hamsters, and macaques
• SC-Ads may be useful mucosal vaccines.
