Mucosal immunization may be important for protection against viral pathogens like HIV, HSV, or RSV. While parenteral immunization can elicit protective immune responses in animal models of these virus infections, effective immunization of humans has been difficult. Each of these pathogens has multiple features that make them difficult vaccine targets (39
), but one feature they share in common is that transmission usually occurs across a mucosal surface. Mucosal immunization against viruses has traditionally been accomplished by using live attenuated viruses. However, for viruses like HIV or HSV that can cause latent or persistent infection, or RSV that infects neonatal airways, the use of replication-competent virus presents a number of safety concerns. Gene-based vector delivery of vaccine antigens affords an option for eliciting immune responses against authentic antigenic structures while avoiding some of the liabilities of the native viral pathogen. However, there have been relatively few options for direct mucosal immunization with gene-based vaccine vectors described, and they have primarily been replication-competent vectors based on adenovirus (40
), picornavirus (41
), rhinovirus (42
), or paramyxovirus (43
). Replication-defective poxvirus and adenovirus vectors have been delivered mucosally to humans, but with limited success (5
). Here we describe the mucosal delivery of DNA facilitated by HPV PsV encapsidation. IVag delivery of HPV PsV-encapsidated DNA induces both T cell and antibody responses to the antigen encoded by the plasmid. HPV PsV encapsidation appears to increase the relative potency of the DNA vaccine based on gene copies delivered, and induces stronger antibody responses than DNA alone.
IVag delivery was more immunogenic than IM. Typically replication-defective gene-based vectors have been less potent when delivered mucosally than when delivered parenterally. The mucosal advantage observed in this report may be due to the natural tropism of HPVs for basal epithelium(7
), but may also reflect access to other antigen presenting cells in the mucosa that are not present in the muscle. Alternatively, there may be selected cells in the mucosa that can recognize the pathogen-associated molecular patterns (PAMPs) in HPV resulting in TLR activation and an adjuvant-like effect that does not happen in the muscle environment. The higher ratio of IgG1 to IgG2a and the reduced ratio of Kd
tetramer responses in the IM immunized group compared to the IVag immunized group suggests that a different pattern of CD4 T cell response was elicited by the two routes. Higher IgG1 is associated with IL-4 production from Th2 responses, and a lower disparity in the epitope hierarchy is associated with Treg responses (38
), and both suggest differences in antigen presentation occurred between IM and IVag administered particles.
Delivery of naked DNA to the disrupted epithelium was also immunogenic, although the copy number of the gene encoding the M/M2 antigen was ~10,000-fold higher in the naked DNA dose than in a dose of HPV-encapsidated plasmids. This and recent in vivo
studies showing improved potency of DNA immunization by electroporation(45
) suggests that the major limitation for DNA vaccination is at the level of delivery into cells and not at one of the many downstream points ultimately required for expression of the vaccine antigen.
IVag delivery of naked DNA was unable to elicit M/M2-specific antibody responses. In contrast, HPV-encapsidated DNA delivered IVag induced antibody responses in the vaginal epithelium and primed for antibody responses in the upper airway. This could involve delivery to alternative targets cells and consequently different antigen processing and presentation pathways, a different pattern of TLR stimulation, or a different threshold of antigen expression needed for T cell vs. antibody induction.
We asked whether the magnitude and kinetics of vaccine antigen availability could explain the distinct properties in the immune response elicited by DNA delivered IVag or IM, either encapsidated with HPV PsV or not. Using luciferase expression as a surrogate for vaccine antigen expression (46
), we found that IVag delivery of DNA by HPV PsV resulted in significantly higher antigen production, but for a brief circumscribed period of time. This transient expression pattern may be attributed to the tropism of HPV targeting basal epithelial cells, which would be expected to differentiate and be sloughed off into the vaginal lumen over the course of about five days. Based on the light emission and RFP expression data, we favor the explanation that IVag HPV PsV improves gene delivery and the magnitude of antigen expression and that antibody is more dose dependent than T cell responses.
IM delivery of the HPV PsV-encapsidated DNA had weak expression in the early days after inoculation. Delivery of naked DNA resulted in better expression when delivered IM than mucosally and luciferase expression was prolonged. However, the level of luciferase expression was much lower than with HPV PsV-encapsidated DNA, and the delayed clearance suggested different target cells were transduced. IVag expressed antigen appears to be immediately available in basal epithelial cells, and based on its rapid clearance, is probably cross-presented by dendritic cells in the mucosa. IM delivered HPV-encapsidated plasmid results in slower more cumulative production of antigen, but the target cell for infection and method of antigen presentation are unclear.
The demonstration of IgA in both nasal and vaginal wash indicates that HPV PsV delivery induced a mucosal antibody response to the antigen encoded by the encapsidated plasmid, and that the vaginal mucosa serves as a local inductive site for the response. In addition to direct induction of IgA in vaginal mucosa there was evidence that IgA responses in nasal secretions were primed by IVag immunization suggesting mobility of the adaptive mucosal response. It was also notable that total IgG was increased in nasal and vaginal washes post-challenge. The evidence of increase transudation at the site of infection and at the site of original immunization suggests that immune effectors were activated at both sites during infection of the airway, and that HPV PsV vector immunization of the vaginal mucosa can induce locally persistent adaptive responses.
HPV PsV-delivered IVag primes for a similar pattern of cytokine response in lung after RSV challenge as other parenterally administered gene-based vectors with no evidence of Th2 cytokines. For RSV in particular and for viral vaccines in general, it is desirable to avoid the induction of Th2 responses. The cells and effector molecules associated with Th2 responses can lead to diminished CD8 T cell function, altered antibody isotypes, and pathology resembling allergic inflammation (47
). This pattern of response has been associated with reduced efficacy and vaccine-induced immunopathology (49
The major limitation of this approach is that disruption of the vaginal epithelium is required for HPV to access its target cells. Nonoxynol-9 (N9) is a licensed, over the counter, spermicide that was used to disrupt the epithelium. It may be clinically acceptable for limited use prior to a vaccination, although repeated use would be unacceptable because of the increased susceptibility to HIV-1 and perhaps other sexually transmitted diseases (50
). In this test-of-concept study, mice were also pretreated with Depo-Provera to thin the vaginal epithelium prior to N9 treatment, and this would not be a clinically acceptable component of a vaccination regimen. Therefore, advancing this concept will require the development of a clinically acceptable approach for providing HPV transient access to its target cells in the basal epithelium.
We have described a vaccine delivery approach using HPV PsV-encapsidated plasmid DNA that results in significant levels of mucosal antigen expression for a brief period of time and is likely to be inducing immune responses primarily through cross-presentation of transduced vaginal epithelium. It has the capacity to induce immunity not only against antigens expressed from the plasmid, but may also provide immunity against the HPV serotype used for the delivery. This is a novel vaccine approach that merits additional investigation especially for pathogens that infect across mucosal surfaces, and particularly for sexually transmitted diseases in women.