This study was designed on the premise that imiquimod and therapeutic HPV vaccination could combine to alter the balance of local immunity through inducing a local inflammatory environment and enhancing T-cell responses to HPV E6 and E7 proteins. Immunotherapies that tip the balance of immune equilibrium in favour of the host effector response and away from regulatory and viral evasion strategies of the HPV may be the key to enhancing the cell-mediated immunity required to eradicate persistent HPV infection and established disease. This study showed that imiquimod followed by vaccination achieved histological clearance of VIN at 52 weeks in almost 60% of a heavily pre-treated cohort of women with high-grade, long-standing VIN.
With the proviso that the study size was small, the analyses of lesion-associated T cells showed that higher pre-existing and post-treatment levels of regulatory T cells are associated with a lack of lesion response to treatment. As expected in the group as a whole, imiquimod treatment induced T-cell infiltration, which was most apparent by week 20. However, the increased CD4 and CD8 T-cell density was significant only in the lesion responder group, whereas a significantly higher regulatory T-cell density was only observed in the non-responder group. These observations are consistent with immune control and therapeutic effect reflecting the balance of useful CD4- and CD8-directed effectors against their control by T regulatory activity. Thus, increasing the CD8 and CD4 T-cell density may be able to re-establish local immune control that is lost or suppressed in chronic VIN (Gul et al, 2004
). Where the T regulatory cells dominate the local immunological milieu, they can continue to suppress the HPV antigen-specific cytotoxic T-cell response facilitating persistent VIN (Kobayashi et al, 2004
; Stanley, 2008
). T regulatory depletion before imiquimod or therapeutic vaccination, particularly in women with higher proportion of systemic or intralesional T regulatory cells before treatment, might enhance the stimulation and efficacy of the useful HPV-specific effector T cells. Indeed, there have been several attempts to improve the outcome of different vaccination regimes either by downregulating or blocking T regulatory cells with anti-CD25 antibodies (Chuang et al, 2009
) or IL-2 diphtheria toxin conjugates (Dannull et al, 2005
The TA-CIN vaccination component of the protocol was aimed at expanding E6- and E7-specific T effectors with the possibility that after imiquimod their entry and activity in the VIN lesions would not be limited by local immunosuppressive factors. Lymphoproliferation of PBMCs established that all patients were immunocompetent and that the vaccination was immunogenic and HPV16 antigen specific. Importantly, these systemic immune responses to HPV16 antigens were significantly associated with lesion responders and not the non-responders. There was no influence on immunity to HPV as a result of the imiquimod treatment. However, a trend towards higher pre-existing responses to HPV16 early antigens in the lesion responder group was noted in this study, which was significantly stimulated by the vaccination. In contrast, the lesion non-responders showed little pre-existing response and this was not boosted by the vaccine. Previous studies have noted a significant correlation between pre-existing systemic HPV16-specific T cells and regression of HPV16-positive lesions (Van Poelgeest et al, 2005
; Winters et al, 2008
). The immune response to the HPV vaccine used in this study might have additional effect if delivered with an adjuvant that can boost both serological and cellular immune responses to its HPV16 antigens (Karanam et al, 2009
Overall, it seems that the natural history of the VIN in the non-responders is related to modulation of both local and systemic immune responses allowing persistence of HPV infection. The mechanisms underlying this chronic VIN state are not known but might include genetic predisposition involving immune and other parameters (Davidson et al, 2003b
). In chronic VIN the suboptimal stimulation of HPV immunity probably also leads to anergy of HPV-specific T effectors. The precise time course of immune responses relevant to HPV lesion clearance are completely unknown and there is no a priori
reason why it would achieve its full potential at the arbitrary study end point especially for chronic lesions, which have been present for many years. Thus, although some of the histological and symptom response noted at week 52 was evident from week 10 (after imiquimod), the response enhanced with time. It is reasonable to argue that an immunologically challenged chronic condition that takes time to establish will equally take time to regress, explaining the continuing response noted long after treatment completion. In this study of a heavily pre-treated cohort of women with long-standing disease, containment of response for 2 years is noteworthy. Patient 5 (40 years, VIN for 20 years and 7 surgical excisions) and patient 19 (35 years, multifocal VIN for 10 years and 10 surgical treatments) illustrate particularly effective lesion responses achieved by week 52 in this study, and that have been maintained for a further 2 years thus far. In both these cases, intralesional pre-treatment T regulatory cell density was low, with no enhancement after treatment, contrary to increased density of CD4 and CD8 cells. A pre-existing proliferative response to HPV16 with significant increase in proliferation response after vaccination was also showed. It would be beneficial if the likelihood of response to treatment could be determined using biomarkers either before treatment or, in the case of combined therapy, after imiquimod to avoid subjecting likely non-responders to unnecessary treatment. At face value, pre-treatment assessment of dominant immunological cell types in the lesion microenvironment and analysing pre-existing immunity to HPV could provide the basis for selecting patients most likely to benefit from imiquimod with or without vaccination regimes.
In considering the imiquimod treatment, tolerability is a significant issue as the majority of women experience local and systemic side effects lasting for the duration of imiquimod treatment, which may affect daily activities. Overall, our regimen was feasible but, as expected, was associated with considerable imiquimod-induced discomfort necessitating breaks in treatment. Women with refractory VIN are, however, highly motivated to comply with the treatment protocol, and in the event, 85% persevered with treatment and finished the full course of imiquimod. In this study, treatment by 8 weeks of imiquimod followed by vaccination gave a lesion response rate of 63% at week 52. This compares very favourably with imiquimod alone treatment of 16 weeks assessed as histological regression to VIN1 or better as 64% soon after treatment (Le et al, 2007
) or with no VIN at 2 months after treatment as 81% (Mathiesen et al, 2007
) or 69% (van Seters et al, 2008
A recent study of VIN patients treated by 3 or 4 immunizations at 3 weekly intervals with a HPV vaccine composed of long HPV16 E6/E7 peptides in adjuvant (Montanide ISA-51, Seppic) showed 60 and 79% lesion size response rate (
50% reduction) at 3 and 12 months of follow-up (Kenter et al, 2009
). As suggested by Kenter et al (2009)
imiquimod might be more beneficial if used after therapeutic HPV vaccination, as studies indicate that imiquimod treatment may depend on IFN-γ
producing HPV-specific T cells.
Following another recently published study of imiquimod followed by PDT (Winters et al, 2008
), continued surveillance for up to 3 years (S Daayana, unpublished results) has shown a sustained clinical response rate of 65% at follow-up. This treatment is now being offered to women in our unit for whom surgical therapy is not suitable. Distinguishing the contribution of the individual components of such combination regimes and establishing proof of either additive or synergistic effects will require further innovative trials. All the recent studies of immunologically driven treatments of VIN provide momentum for further multicentre randomised trials with consistency in measurement of outcomes and definitions of response. Comparing upfront surgical treatment with imiquimod or other potentially more potent TLR agonists (Fahey et al, 2009
) or therapeutic HPV vaccination (Karanam et al, 2009
; Kenter et al, 2009
), or a combination of TLR agonists with therapeutic HPV vaccination in a crossover study design will be valuable.