1. Volume 8, Issue 3, Pages 392-399 (September 2003)
Systemic and mucosal antibody responses following retroductal gene transfer to the salivary gland 
Sean N Tucker, Karen Lin, Sasha Stevens, Roland Scollay, Michael J Bennett, David C Olson 
Molecular Therapy 
Volume 8, Issue 3, Pages (September 2003)
DOI: /S (03)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Antibody responses following salivary gland DNA administration. (A) IgG and IgA isotypes were measured 3 weeks after 175 μg of plasmid DNA encoding hGH was infused retroductally into each salivary gland (SG) or injected intramuscularly (im) into each leg. As a control, 15 μg of hGH protein was injected sc in a 50/50 mixture of sterile saline/Freund's complete adjuvant (Protein + CFA). By Mann–Whitney, SG exceeds im (P < 0.03 for both IgG and IgA titers). (B) Plasma IgG titers were examined over time following DNA administration. In this experiment, submucosal refers to needle injection into the tissue below the tongue. Salivary gland, intramuscular, and submucosal groups were given one dose of 175 μg DNA per rat at the time shown by the arrow. Statistical analysis by Mann–Whitney demonstrated that SG is better than submucosal (P = 0.01) and better than im (P < 0.01). Error bars represent the SEM.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Antibody responses to gp120 following salivary gland DNA administration. (A) Antibody responses were measured through time. Arrows indicate when 88 μg of plasmid DNA was retroductally infused into each salivary gland (SG) or injected into each leg (im). SG-treated animals used a 3.6 mM Zn formulation. At completion of the experiment, the antibody titers were measured before and 2 weeks after re-treatment to look for memory-type responses (P < 0.05 for comparison between im and SG groups at maximum titer time points (weeks 11 and 39). (B) Anti-gp120 IgG titers were measured over time. In this experiment, different formulations were evaluated for the ability to elicit IgG antibodies following RSGV. These include DNA coformulated with Lipid/Zn, Zn alone, or water or left untreated [19,20]. Arrows represent the times the treated animals were given DNA. Zn was better than water by Mann–Whitney at 3 weeks (P < 0.03), but indistinguishable from water at 6 weeks (P = 0.7).
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Mucosal immune responses following salivary gland DNA vaccination. (A) Anti-gp120 IgA and IgG were measured by ELISA. Salivary gland-vaccinated animals were compared to untreated animals at 6 weeks post-initial vaccination for their fecal IgA response and saliva IgA response to gp120. Saliva demonstrated statistically significant responses compared to untreated (P = 0.02 by Student's t test). In a separate experiment using female rats, specific IgG was compared after normalizing to use equivalent amounts of IgG. Salivary gland-treated animals produced significant vaginal IgG responses (N = 4) compared to the untreated animals (P = 0.02). (B) Cells were isolated from Peyer's patches from a variety of vaccine groups. The isolated cells were placed in ELISPOT plates coated with gp120 protein to measure the numbers of antibody-secreting cells (ASC) that recognize the antigen. SG was better than im for producing Peyer's patch ASC by Student's t test (P = 0.03). (C) Lung lavages were analyzed for specific antibodies to gp weeks after the last salivary gland vaccination (SG). Samples were normalized to 25 ng/ml IgA (for measurement of specific IgA and secretory component (S.C.)) and 100 ng/ml total IgG before an OD value was determined by ELISA. IgA and secretory-specific responses were statistically significant (P = 0.046, P = 0.02, respectively).
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Antibody responses to anthrax PA. (A) Antibody titers were measured following retroductal delivery of PA DNA to the salivary gland (SG/PA DNA), injection of PA DNA into the muscle (i.m./PA DNA), or retroductal delivery of an irrelevant DNA to the salivary gland (SG/hGH DNA). Subcutaneous PA protein plus CFA vaccination (s.c./Prtn + CFA) and naive animals served as positive and negative controls, respectively. Arrows indicate when DNA or protein was administered. (B) Antibody responses to PA within lung lavages were measured 2 weeks after lethal anthrax toxin challenge. Samples were normalized to use three different equivalent amounts of total IgA (50, 17, and 1.9 ng/ml) before measurement.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions