In the present work, we studied HPV16 E2 protein because of its early and strong expression 
. E2 is a larger protein than E6 and E7 proteins and it induces more T-cell responses than E6 and E7 
. Therefore looking for an E2-specific response appeared to be more sensitive. In addition, E2 is required for viral replication 
and the detection of E2-specific T cell responses is the signature of this event 
. The study of anti-E2 T-cell responses is thus appropriate in the early phases of HPV16 infection or for pre-malignant lesions as usual VIN. We observed proliferative responses to HPV16-E2 antigens in 6 out of 8 women presenting with usual HPV16-associated VIN. Two of them were asymptomatic at study entry (treated 3 and 4 months before by laser or electrocoagulation, respectively) whereas in the four other women proliferative responses strongly linked with ongoing clearance of the lesion after treatment (imiquimod or laser). In contrast, no E2-specific responses were detected in patients with persistent lesions and in the four other ones before they underwent VIN treatment. Then the association between the disappearance of the HPV-16 induced vulvar lesions under therapy and the development of E2-specific proliferative T cell responses is striking. The appearance of anti-E2 proliferative T-cell responses could be a consequence of the release of E2 protein after local therapy, as they did not occur spontaneously.
The six women who demonstrated proliferative responses also had CD4+ T-cells synthesizing IL2 alone, IFNγ alone or both IFNγ and IL2. Such a profile (presence of proliferating and cytokine-producing polyfunctional population) suggests that these CD4+ T-cell responses are anti-E2 memory CD4+ T-cell responses as previously described by Harari 
Moreover, all women, whether they cleared or not their lesion after treatment, had very high number of anti-HPV16-E2 T-cells as tested by ex vivo
IFNγ ELISPOT. The presence of a monofunctional population producing IFNγ only, without detectable proliferation observed in the women presenting with persistent lesions (F#1, #7 and #3, #5, #6, #8 before treatment) is consistent with the presence of effector T-cells and an uncontrolled HPV16 infection. This is in agreement with previous study showing that the detection of specific immune responses against E2 peptides by IFNγ ELISPOT did not correlate with either HPV16 control or VIN or CIN1 clearance 
. Moreover, after local therapy and healing, the number of IFNγ-producing T-cells decreased, as observed in women F#3 and F#8. The latter observation suggests that the monofunctional T-cell population producing IFNγ only depends on the quantity of HPV16-E2 protein synthesized.
Strong anti-E2 IFNγ ELISPOT responses against HPV16 309–323 E2 peptide were found in all women. This 15-mer peptide, included in the E2-10 peptides pool, shares more than 70% amino acids (by BLAST analysis) only with cutaneous HPV2 and 27 and with mucosal HPV55. Five of eight women had a past history of common warts (F#1, #4, #5, #6, #7) (). In order to exclude a possible cross-reactivity between HPV types, we performed ex vivo IFNγ ELISPOT assays against E2 309–323 peptide from HPV2, 27 and 55 that were all negative (). Consequently, we conclude that the IFNγ ELISPOT responses against HPV16 309–323 E2 peptide are indeed specific for HPV16.
Ex vivo IFNγ ELISPOT responses in women and men against HPV16, HPV2, HPV27 and HPV55 E2 309–323 peptide.
In the absence of condom usage for at least 6 months, the male partners of the eight women could be contaminated by HPV16. HPV16 and HPV27 (a cutaneous HPV) were identified in genital sampling gathered by cytobrushing in only two of the eight healthy partners (M#8, #5) (). Such a prevalence of HPV16 contamination is similar to the one usually observed in male partners of oncogenic HPV-infected women 
. This does not exclude the possibility that the other male partners could also be infected either at undetectable levels or in anatomical sites not sampled by the cytobrush.
We also observed HPV16-E2-specific proliferative responses in seven male partners and intracellular synthesis of single IFNγ, dual IFNγ/IL2 and single IL2 in six of them. These E2 specific T-cell responses in all male partners but two (M#4, #5) indicate a striking link between the absence of HPV-related lesions and the presence of spontaneous E2-specific proliferative T-cell responses and single IFNγ, dual IFNγ/IL2, single IL2 T-cell production, as previously described in other viral systems 
. Such a spontaneous polyfunctional anti-E2 T-cell response could be due to an efficient presentation of viral antigens by dendritic cells present in mucosal tissue and it is tempting to speculate that E2-specific responses prevent HPV16-related lesions. Therefore, spontaneous HPV16 control could be related to the presence of polyfunctional memory CD4+ T-cells in male partners.
Peptide 309–323 was also the dominant peptide detected in IFNγ ELISPOT assay in male partners. Indeed, high frequencies (>500 SFC/106
PBMC) of blood effector T-cells, directed against E2 309–323 peptide and synthesizing IFNγ alone without proliferative capacity were identified in the majority of male partners (6/8) by ex vivo
anti-E2 peptide IFNγ ELISPOT assays. The level of this response with very high number of spots is similar to that observed during the chronic phase of HIV infection characterized by highly replicative viral reactivation 
. In the six men tested, there was no detectable cross reactivity with 309–323 E2 peptide from HPV2, 22 and 55, similarly to the findings in women (). Since E2 protein is not encapsidated, stimulation of anti-E2 cellular immune responses is a mark of the presence of viral replication in infected cells. The high frequency of E2-specific T-cells measured in partners of women with usual VIN demonstrates that the virus effectively replicates in males.
In summary, the analysis of E2-specific T-cell responses is a sensitive and reliable tool to analyze disease progression and the natural history of HPV infection and premalignant lesions. In women, polyfunctional T-cell responses featured by proliferative responses and cytokine synthesis reflect memory CD4+ T-cell population induced by VIN treatment. Monofunctional T-cell responses restricted to ex vivo IFNγ ELISPOT reflect the presence of numerous HPV16 E2-specific effector T-cells but does not correlate with control of HPV16 infection.
In the male partners, the presence of polyfunctional spontaneous T-cell proliferative responses with single IL2, dual IFNγ/IL2, and single IFNγ memory T-cells against HPV16 E2 peptides in the absence of detectable lesion despite HPV16 exposure suggests an efficient and spontaneous control of genital HPV lesions. These results are reminiscent of those described in Gambian prostitutes who, despite frequent exposure to HIV showed strong anti-HIV cytotoxic T-cell responses and remained uninfected 
. Such anti-HPV16 T cells responses may thus reflect the control of viral infection.
We therefore demonstrate that, although not clinically detectable, HPV16 can replicate in men and induce a strong memory T cell response against one of the early viral proteins. Despite the fact that they were obtained in a small cohort of couples, our results suggest that males are an important reservoir of genital HPVs and provide an argument in favor of prophylactic HPV vaccination of young men with virus like particles in order to prevent HPV16 infection in men and thus fight against the spread of mucosal HPV diseases in the population.