3.1. Migration of Ad5 and HSV-1 virus particles in whole saliva
Purified, fluorescent Ad5 and HSV-1 virus particles were suspended in whole saliva or water, and then loaded into custom-made microfluidic chambers in order to analyze their mobility, using fluorescent videomicroscopy. Trajectories were captured from multiple 15 second long movies for each condition for an average of 70 individual particles per condition. Two representative trajectories for HSV-1 and Ad5 virus particles in saliva are presented in , which shows that the Ad5 particle was almost completely immobilized in saliva, while the HSV-1 virion diffused freely.
Adenovirus particles, but not HSV-1 virions, are trapped in saliva
The trajectories were used to calculate the mean squared displacements (MSD) as a function of time for each condition. These results are presented in . Consistent with their smaller size (90 nm diameter for adenovirus versus 180 nm for HSV; [35
]), Ad5 particles exhibited considerably greater mobility in water than HSV-1 virions. However, when Ad5 particles were resuspended in saliva, their mobility was greatly reduced when compared to water (; p=0.0028, Mann Whitney test). In contrast, HSV-1 virions showed roughly equivalent mobility in either water or saliva.
The diffusion coefficient of the entire virus population analyzed was also calculated, in both water and saliva. The ratio of the diffusion coefficients of Ad5 in saliva compared to water (Ds/Dw) was found to be 0.048, indicating that virus mobility in saliva was reduced by approximately 20-fold compared to its mobility in water. In contrast, the ratio of the diffusion coefficients of HSV-1 in saliva compared to water (Ds/Dw) was 0.93, indicating that virus mobility in saliva was essentially equivalent to its mobility in water (). The difference in the Ds/Dw ratio for Ad versus HSV virions was found to be statistically significant (p<0.0001, unpaired t test).
3.2 Penetration of sublingual epithelium by rAd5 and HSV-1 amplicon virus vectors
We next examined the ability of rAd5 and HSV-1 amplicon vectors to drive expression of an encoded transgene following sublingual delivery in a mouse. To do this, an Ad5 vector expressing firefly luciferase (rAd5:Luc) and a helper-free (hf) HSV-1 amplicon vector encoding the same gene (HSV-1:Luc) were delivered via the SL and IM route to BALB/c mice. Expression of the encoded luciferase transgene was then measured 24 hours later, using In Vivo Imaging System (IVIS) technology.
For this experiment, we selected viral inocula that were expected to yield similar levels of gene expression following delivery by the IM route. Thus, we used 1×108 viral particles (VP) for the Ad5:Luc vector and 5×105 VP for HSV-1:Luc. As expected, IM delivery of both vectors resulted in similar, strong levels of luciferase expression in vivo (1.23±0.09 × 107 photons/second [p/s] and 2.66±0.63 × 107 p/s, respectively) (). However, SL delivery of the HSV-1:Luc elicited very low levels of luciferase expression, suggesting that this vector was unable to efficiently penetrate the murine sublingual epithelium (). In contrast, SL delivery of Ad5:Luc resulted in strong luciferase expression, indicating that the vector was able to effectively penetrate the sublingual epithelium (3.63±1.73 × 106 p/s) (). As noted in , the difference in the luciferase expression levels for sublingually delivered Ad5:Luc versus sublingually delivered HSV-1:Luc achieved statistical significance (p=0.0286, Mann Whitney test).
An Ad5 encoding firefly luciferase efficiently penetrates the sublingual mucosa following SL instillation, while a HSV-1 amplicon vector encoding a matched insert fails to do so
It can be readily appreciated that the absolute magnitude of measured luciferase expression was considerably greater in animals that received the rAd5:luc vector via the IM route, compared to those that received the same dose of vector via the SL route (). The significance of this observation is unclear, however, since in vivo analysis of luciferase expression at the SL site of delivery necessarily requires imaging through the soft palate – likely resulting in diminution of signal intensity. In contrast, muscle tissue can be imaged directly, and may therefore tend to yield higher apparent levels of luciferase activity. Regardless, the data show that the rAd5 vector, but not the hf HSV-1 amplicon vector, was able to effectively penetrate the sublingual epithelium.
3.3 Saliva does not attenuate HSV-1 infectivity
One possible explanation for the very poor gene expression efficiency by sublingually delivered HSV-1:Luc is that the vector may be inactivated by saliva. We therefore tested whether incubation of HSV-1:Luc with whole saliva resulted in a loss of vector infectivity in vitro. For this experiment, HSV-1:Luc vector was incubated with saliva samples from four different human subjects, and then added to HEK 293A target cells. Twenty-four hours later, the cells were collected and lysed, and luciferase activity in the cell lysates was determined.
Both vectors efficiently expressed the reporter gene in HEK293A cells. Luciferase expression levels in cells exposed to control virus stocks (not incubated with saliva) was assigned a level of 100%, and luciferase levels in cells exposed to saliva-treated virus stocks was calculated as a percentage of this baseline. As shown in , incubation with unstimulated whole human saliva had no effect on the infectivity of the HSV-1:Luc vector (99% of baseline expression).
We also conducted an analogous experiment using whole, pilocarpine-stimulated mouse saliva (pilocarpine stimulation being necessary to generate a sufficient volume of saliva to perform the study). In this experiment, a HSV-1 amplicon vector encoding a green fluorescent protein (GFP) reporter gene was used, because available HSV-1:Luc stocks had been depleted. The HSV-GFP vector efficiently transduced HEK293A cells. The percentage of transduced (GFP positive cells) in cultures exposed to control virus stocks (not incubated with saliva) was assigned a level of 100%, and the percentage of transduced cells in cultures exposed to saliva-treated virus stocks was then calculated as a percentage of this baseline. As shown in , incubation with pilocarpine-stimulated whole mouse saliva had no effect on the infectivity of the HSV-1:GFP vector (96% of baseline expression). We conclude that the very poor in vivo gene expression efficiency by sublingually delivered HSV-1:Luc () cannot be attributed to virus inactivation by saliva.
3.4 Humoral immune response to sublingual delivery of rAd5 and hf HSV-1 amplicon vectors encoding HIV-1 gp120
rAd5 and hf HSV-1 vectors encoding an identical human codon-optimized HIV-1 Env gene (HIV-1MN gp120) were delivered to BALB/c mice via either the SL or IM route, using a homologous prime-boost immunization regimen. Animals were boosted on day 21, and sacrificed on day 31. Sera were then collected and HIV-1 Env-specific antibody responses were measured by IgA and IgG ELISA assay.
IM delivery of both vectors elicited a robust Env-specific IgG response in the serum of immunized mice (). In contrast, only rAd:gp120 elicited a strong Env-specific IgG response when delivered by the SL route (). The difference in the magnitude of the Env-specific IgG response elicited by sublingual delivery of rAd:gp120 versus HSV-1:gp120 achieved statistical significance at all three serum dilutions tested (p=0.0286; Mann-Whitney test).
Sublingual administration of an Ad5 vector expressing HIV-1 gp120 elicits robust antigen-specific serum IgG and IgA immune responses, while sublingual administration of a matched HSV-1 amplicon vector fails to do so
The Env-specific serum IgA response was also measured in these animals. SL administration of rAd5:gp120 elicited a robust HIV-1 Env specific IgA response in serum, but all other immunization routes/vectors failed to do so (including SL delivery of the HSV:gp120 vector and IM delivery of the rAd5:gp120 and HSV:gp120 vectors) (). The difference in the magnitude of the Env-specific IgA response elicited by sublingual delivery of rAd:gp120 versus HSV-1:gp120 achieved statistical significance at the 1:100 serum dilution level (p=0.0286; Mann-Whitney test), as indicated.
3.5 Robust serum and mucosal IgG and IgA antibody responses to sublingual delivery of an rAd5 vector encoding oligomeric HIV-1 Env
In order to confirm the reproducibility of the results shown in , we constructed an E1-deleted, replication-defective rAd5 vector encoding oligomeric HIV-1 Env gp140 (YU2 strain). This vector (rAd5:gp140) was administered to BALB/c mice via the IM, SL or intra-gastric (IG) route. A homologous prime-boost regimen was employed, with a boost at day 28; the animals were sacrificed and samples taken for analysis on day 38. IM or SL delivery of the rAd5:gp140 vector elicited equivalent levels of Env-binding serum IgG antibodies (). In contrast, SL delivery, but not IM delivery, of the rAd5:gp140 vector resulted in a robust HIV-1 Env specific serum IgA response () - confirming the data generated with the rAd5:gp120 vector ().
Sublingual delivery of an Ad5 vector expressing oligomeric HIV-1 Env (gp140) induces robust, antigen-specific IgG and IgA antibody responses in both serum and in vaginal washes
IG delivery of the rAd5:gp140 vector elicited a considerably weaker Env-specific IgG and IgA serum antibody response, compared with animals that received the same dose of vector via the SL route (). These data suggest that swallowing of the rAd5:gp140 vector likely cannot account for the robust HIV-1 specific humoral response elicited by SL vector delivery.
In this experiment, vaginal wash samples were also collected at the time of sacrifice from mice that received the rAd5:gp140 vector via the SL or IM route, as well as from control mice (which received PBS). Env-specific IgG and IgA antibody responses were detected in vaginal washes from animals that received the rAd5:gp140 vector via the SL route ( respectively). IM administration of the same vector dose resulted in reduced HIV-1 Env specific IgG responses in the vaginal wash, and undetectable Env-specific IgA responses.
3.6 High-dose sublingual delivery of an Ad5 vector expressing oligomeric HIV-1 Env (gp140) results in enhanced antigen-specific IgA antibody responses in vaginal washes
The data shown in revealed a trend towards enhanced Env-specific, vaginal IgA and IgG responses following SL delivery of the rAd5:gp140 vector. Based on these encouraging findings, we performed a followup experiment using a higher dose of the same vector (2×109 VP, or 20-fold higher than the dose used in the experiment shown in ) and a second boost (i.e., boosts at both day 21 and 42 following initial priming). As shown in , this intensified immunization regimen revealed a statistically significant difference in the level of Env-specific IgA in vaginal wash samples from mice immunized with Ad:gp140 via the SL route versus mice immunized via the IM route (p=0.0079, Mann-Whitney test).
High-dose sublingual delivery of an Ad5 vector expressing oligomeric HIV-1 Env (gp140) results in enhanced antigen-specific IgA antibody responses in vaginal washes
3.7 Quantitation of Env-specific antigen secreting cells and neutralizing antibodies elicited in response to sublingual delivery of a rAd5 vector encoding oligomeric HIV-1 Env
In order to quantitate the magnitude of the B cell response elicited by sublingual delivery of an Env-encoding Ad5 vector, rAd5:gp140 particles were delivered to BALB/c via the SL or IM route at day 0, followed by a homologous boost at days 28 and 56 via the same route. On day 63 (seven days after the final boost), single-cell splenocyte suspensions were prepared and Env-specific antibody secreting cells (ASC) were quantitated using an HIV-1 Env (YU2) specific ELIspot assay. SL and IM delivery of the rAd5:gp140 vector elicited approximately equivalent frequencies of HIV-1 Env specific IgG ASC in the spleens of immunized animals (23.0 +/− 2.4 versus 16.0 +/− 5.5 Env-specific ASC per 106 splenocytes in animals that received vector by the SL and IM routes, respectively) (). In contrast, only SL vector delivery elicited HIV-1 specific IgA ASC (40.0 +/− 19.5 versus 2.0 +/− 1.4 Env-specific ASC per 106 splenocytes in animals that received vector via the SL and IM routes, respectively). Statistical data analysis confirmed that the difference in the magnitude of the Env-specific IgA ASC response elicited by SL versus IM delivery of rAd5:gp140 achieved statistical significance (p=0.0286; Mann-Whitney test).
Sublingual delivery of an Ad5 vector expressing oligomeric HIV-1 Env (gp140) elicits Env-specific IgG and IgA antibody secreting cells (ASC) in spleens of immunized mice
In addition to quantifying Env-specific ASC, Env-specific neutralizing antibody (Nab) titers were also measured using two clade B strains of HIV-1 (YU2 and SF162.LS). This analysis showed that SL vaccine delivery elicited serum neutralizing antibodies against one or both of the HIV-1 strains tested in 3 of 3 mice that were analyzed, while IM vector delivery elicited detectable Nabs in 2 of 3 animals tested ().
Neutralizing antibody titers from mice immunized with rAd5:gp140 vector.