The cross-protective efficacy of the HPV-16/18 vaccine against HPV-31/33/45 was demonstrated in an earlier large clinical study (HPV-008)17,23,24
in a cohort of women who were HPV DNA-negative for corresponding HPV type at baseline, regardless of serostatus.17
In the total vaccinated cohort for efficacy, vaccine efficacy against 6 mo persistent infection attributed to HPV-31, -33 and -45 increased over time from an interim analysis, performed at a mean follow-up of 14.8 mo [standard deviation (SD): 4.9 mo] after third vaccine dose (36.1%, 36.5% and 59.9%, respectively),24
through to analysis at 34.9 mo (SD: 6.4 mo) (66.9%, 42.2% and 71.6%, respectively).17
Recent results from the end-of-study analysis of this trial confirmed the cross-protective efficacy of the HPV-16/18 vaccine against these three HPV types through to Month 48.23
Together, HPV types -16, -18, -31, -33 and -45 account for approximately 82% of cervical cancers.25
Cross-protection results have also been published for the HPV-6/11/16/18 vaccine,20
although it should be noted that these should not be directly compared with the HPV-16/18 results, as the study designs of the HPV-16/18 vaccine and HPV-6/11/16/18 vaccine trials differ in HPV DNA- and immunological assays, endpoints and study populations. In a cohort of women who were seronegative and DNA-negative for HPV types in the HPV-6/11/16/18 vaccine, and DNA-negative for ten non-vaccine HPV types (including HPV-31/45), the HPV-6/11/16/18 vaccine demonstrated cross-protective efficacy against CIN1.3 or adenocarcinoma in situ associated with HPV-31; no cross-protection was shown against CIN1.3 or adenocarcinoma in situ associated with HPV-45.20
The current study was designed to directly compare the immune response to non-vaccine oncogenic HPV types elicited by the HPV-16/18 vaccine and the HPV-6/11/16/18 vaccine. Our sub-analysis of the HPV-010 study, along with the available data generated in clinical efficacy studies, was intended to provide insight into the potential mechanism(s) for cross-protection.
Our observations show that both the HPV-16/18 vaccine and the HPV-6/11/16/18 vaccine induced cross-reactive serologic responses against HPV-31 and HPV-45. Humoral immune responses for non-vaccine types HPV-31/45 (PBNA and ELISA) were much lower than those for vaccine types HPV-16/18.21,22
At Month 24, there were no significant differences between the HPV-16/18 vaccine and the HPV-6/11/16/18 vaccine in terms of HPV-31/45 circulating antibodies as measured by PBNA. It should be noted that circulating neutralizing antibodies were at levels close to or below the limit of detection of each assay. Similar levels of circulating antibodies (measured by ELISA) were observed between both vaccines in the 27–35 y and 36–45 y groups; for both vaccines, the highest levels of HPV-31 and HPV-45 circulating antibodies were reported in the 18–26 y group.
Previous results from the HPV-010 study demonstrated higher vaccine specific immune responses with the HPV-16/18 vaccine compared with the HPV-6/11/16/18 vaccine, as supported by higher levels of specific neutralizing antibodies, seropositivity rates and a higher frequency of HPV cross-reactive memory B cells and T cells.21,22
However, in our sub-analysis, the levels of neutralizing antibodies to HPV-31/45 were relatively low for both vaccines. There was also no significant difference in circulating HPV cross-reactive memory B-cell response for HPV-31 and HPV-45 with both vaccines, likely due to the fact that most memory B cells would have migrated to central lymphoid organs, such as the spleen, to be subsequently activated by the interaction of circulating antigen presenting cells such as CD4+ T cells.26
An earlier study from women immunized with the HPV-6/11/16/18 vaccine, investigating vaccine-induced antibody binding and neutralization with HPV-18/45 using PBNA, also demonstrated relatively low levels of vaccine-induced antibodies to cross-neutralize HPV-45 pseudovirions.27
Findings from a recent study by Kemp et al. suggest that low levels of cross-neutralizing antibodies may contribute to the mechanism of cross-protection observed with the HPV-16/18 vaccine.28
It may simply follow that the response to HPV-16 and -18 is higher due to the immunodominance of the type-specific epitopes, so only a relatively small proportion are shared with phylogenetic family members like HPV-31 and -45, respectively.
A separate study showed that a fourth dose of the HPV-16/18 vaccine induced an anamnestic response (evidenced by a rapid and strong increase of antibody titers) not only to the vaccine types HPV-16/18, but also to non-vaccine types HPV-31/45.29
The demonstration of immunologic memory for the HPV-31/45 response in women confirms that the HPV-16/18 vaccine does induce cross-reactive immune responses even though circulating neutralizing cross-reactive antibodies are at levels close to or below the limit of detection of the assays.30
The main immunological parameter that differentiates the immune response to HPV-31/45 is the CD4+ T-cell response. In our sub-analysis, CD4+ T-cell responses remained high from Month 7 to Month 24 and the HPV-16/18 vaccine gave higher CD4+ T-cell responses than the HPV-6/11/16/18 vaccine at all timepoints. Importantly, in contrast to the relatively low levels of cross-reactive antibodies, the frequency and quality of the crossreactive CD4+ T-cell responses to HPV-31 and -45 were similar to the specific responses to HPV-16 and -18.22
However, we note that, based on published data,17,20
there appear to be differences in the HPV types for which the vaccines confer cross-protective efficacy. This suggests that higher levels of CD4+
T-cell response may be necessary for cross-protection against certain HPV types, such as HPV-45, but that the requirements for cross-protection against other types such as HPV-31 might be different.
Of note, the cross-reactive T-cell response to HPV-31 and -45 was measured using pools of synthetic peptides spanning the truncated VLP L1 sequences of HPV-31 and HPV-45. We cannot exclude the possibility that the HPV-6/11/16/18 vaccine induced a cross-reactive T-cell response against the portion absent (approximately 30 amino acids) from the truncated HPV-31/45 L1 VLPs. However, such bias is unlikely given that the truncated portion is small and also the similarity of the HPV-31/45 T-cell responses to the specific responses to HPV-16/18 (obtained using a pool of peptides spanning the entire VLP L1 sequences of HPV-16/18).
Other assays used in the current study are not anticipated to have introduced a bias toward either vaccine. As discussed previously in references 21
, no bias was observed for HPV-16/18 data obtained using the ELISA or memory B-cell data obtained using the ELISPOT assay. For the PBNA used in the present study, the amino acid sequence of the L1 protein and the cell-line used for the production of the pseudovirions, which contain L1 and L2, are different from those used in either vaccine. Importantly, PBNA and ELISA have also been shown to correlate with Merck's competitive Luminex immunoassay (cLIA).31,32
Moreover, none of the assays, nor the HPV-16/18 vaccine or the HPV-6/11/16/18 vaccine contain HPV-31/45 VLPs.
Adjuvant systems have been shown to enhance specific and cross-clade neutralizing antibody immunological responses in addition to T-cell responses.33–35
In the case of the HPV-16/18 vaccine, the AS04 adjuvant system,36
which contains MPL (50 µg) absorbed on aluminum salt (Al3+
, 500 µg), may similarly enhance the immune responses. The AS04 component of the HPV-16/18 vaccine was shown to induce a higher frequency of type-specific memory B cells and antibody responses against HPV-16/18 compared with the same vaccine formulated with aluminum salt alone.8
The MPL component of the AS04 adjuvant binds and activates the Toll-like receptor-4 (TLR-4), which is present on key antigen-presenting cells that play an important role in the induction of innate and adaptive immune responses.37,38
Furthermore, the combination of an aluminum salt with MPL is thought to prolong cytokine responses at the injection site.36
Taken together, these factors may plausibly account for the higher CD4+ T-cell response against HPV-31/45 observed with the HPV-16/18 vaccine compared with the HPV-6/11/16/18 vaccine in this study.
Although the mechanism(s) of cross-protection has not yet been fully elucidated, it is likely to be associated with the phylogenetic relationship of HPV types. HPV-16, -18, -31 and -45 belong to the genus α-papillomavirus, which is further classified by species and then type; the A7 species includes HPV types -18 and -45 and the A9 species includes HPV types -16 and -31 (). HPV types belonging to the same species are phylogenetically related;39,40
based on predicted L1 amino acid sequences, HPV-31 shares 83% homology with the L1 protein of HPV-16 and HPV-45 shares 88% homology with the L1 protein of HPV-18.20
The observed difference in the cross-protective efficacy for HPV-45 between the vaccines is probably not related to the primary sequence of the HPV-18 L1 VLPs, since both share a similar percentage of sequence homology with HPV-45. However, the truncation of ~30 amino acids at the C-terminus of the HPV-16/18 vaccine's HPV-18 L1 VLPs41
might impact on the accessibility of epitopes that are shared between HPV-18 and HPV-45.
Figure 9 Phylogenetic tree of anogenital human papillomavirus types (adapted from Schiffman and Wentzensen 2010 and Schiffman et al. 2005).54,55 This phylogenetic tree is based on the alignment of concatenated early and late open reading frames. The carcinogenicity (more ...)
Confirmation would require further biophysical structural characterization of HPV-18 L1 VLPs, the demonstration of differences in epitope recognition by antibodies induced by the two vaccines, or evaluation of the relative avidity of antibodies for the different epitopes. In addition, conformational differences in L1 VLP epitope exposure due to differences in vaccine production process and/or the adjuvant formulation (as discussed above) may also contribute to differences in the quality of cross-reactive neutralizing antibody responses, and may contribute to the differences in cross-protection observed between the vaccines.
Despite high levels of homology between HPV types of the same species, even if the conformational structures of L1 VLPs from different HPV types are very similar, the surface loops that contain neutralizing domains display significant amino acid heterogeneity. The unique features of these surface loops are the distinct surface immunodominant conformational epitopes that provide type-specific protection. There are high levels of conserved homology between HPV types that may represent subdominant cross-reactive epitopes.42
As cross-neutralization induced by L1 VLPs represents less than 1% of the type specific neutralizing activity induced by the immunodominant conformational epitopes, it is uncertain whether this is sufficient to offer cross-protection in vivo.43
The capacity of L1-VLP-induced antibodies to mediate type-specific and cross-protection against cervicovaginal challenge was recently demonstrated in a murine challenge model, following vaccination with HPV-16 VLPs (on alum) or following passive transfer of immune serum.44
A high level of specific protection was observed against HPV-16 and partial cross-protection was observed against HPV-31 challenge.44
Furthermore, in rabbits that received the HPV-16/18 vaccine, high levels of cross-protection against HPV-31/45 were observed after HPV quasivirion [virions with human HPV L1/L2 capsids and the cotton-tail rabbit papillomavirus (CRPV) genome, produced in 293TT cells] 45
challenge in the presence of no or low levels of neutralizing antibodies.46
Importantly these controlled experimental studies clearly demonstrate the capacity of L1 VLPs to induce cross-protective immunity and indicate that the cross-protection observed in clinical studies resulted from vaccine-induced immune responses.
Investigations into the mechanism of L1 VLP-induced prevention of HPV infection have led to the proposal of two distinct mechanisms of protection by L1 specific polyclonal antibodies: (1) high levels of antibodies result in an immunoglobulin-coated HPV capsid, which prevents interaction of the capsid with the cell surface, (2) a lower antibody level allows the capsid to associate with the cell surface but prevents the conformational changes required for virus entry.44,47
It has been suggested that cross-protection and type-specific long-term protection may be attributed to the second mechanism with a relatively low antibody to virus ratio and that PBNA may not be a suitable assay to assess cross neutralizing antibodies.44
The National Cancer Institute (United States) is currently investigating the possibility of developing more accurate methods to assess all sets of neutralizing antibodies.47
An alternative role of antibodies in mediating cross-protection follows from the observation that HPV suppresses Langerhans cell activation, resulting in local immune suppression. Interestingly, this suppression can be reversed when Langerhans cells encounter HPV virions or L1/L2 VLPs in the presence of antibodies.48,49
Thus, HPV infection in the presence of vaccine-induced antibodies could activate Langerhans cells, via the Fc receptor or other mechanisms, and lead to local inflammation that does not occur in the absence of antibodies. Importantly, the antibodies would not need to have the functional capacity to neutralize the virus.
Furthermore, a role for CD4+ T-cells in cross-protection in the presence of cross-reactive antibodies is proposed. It has been shown that CD4+ T-cells can activate NK cells in the presence of pro-inflammatory signals,50
suggesting that the combination of activated Langerhans cells, due to the presence of antibodies,46
and elevated frequencies of cross-reactive cytokine producing CD4+ T-cells could activate IFNγ producing NK cell responses.48
Thus both CD4+ T- and NK cells could contribute to cytokine-mediated reduction of viral replication and/or elimination of viral infected epithelial cells.
Assuming that CD4+ T-cells and NK cells have a role to play, one would expect to see better cross-protection for progressive disease efficacy endpoints such as CIN2+/3+, due to elimination of infected cells, than for incident/persistent infection efficacy parameters. Although there is some evidence of this pattern being observed from AS04-adjuvanted HPV-16/18 vaccine efficacy studies, it is not unequivocal and further investigation will provide more complete data to verify this hypothesis.17
Notably, CD4+ T-cell responses have been shown to be important in limiting the progression of cervical cancerous lesions51
and in the regression of genital warts.52
In a study examining the efficacy of a novel therapeutic vaccine containing HPV-16 E6 and/or E7 synthetic peptides, 60% (95% CI: 36–81) of patients with HPV-16-positive vulvar intraepithelial neoplasia had a clinical response three months after last vaccination; patients with a complete response had a significantly higher CD4+ T-cell response than patients without a complete response.51
Therefore, a CD4+ T-cell-mediated mechanism that eliminates HPV-infected cells is plausible.
However, our study assessed only immunological endpoints; whether or not the higher levels of cross-reactive CD4+ T cells observed with the HPV-16/18 vaccine compared with the HPV-6/11/16/18 vaccine correlate with enhanced protection against the progression of cervical lesions has not been assessed.
In conclusion, vaccination with the HPV-16/18 vaccine or the HPV-6/11/16/18 vaccine induces humoral responses to non-vaccine HPV types -31/45, albeit at generally low levels; in the case of the HPV-16/18 vaccine, this response has demonstrated immunological memory in other investigations. The HPV-16/18 vaccine induced relatively higher HPV-31/45-specific CD4+ T-cell responses compared with the HPV-6/11/16/18 vaccine, which may play a role in cross-protection; however, further studies are necessary to fully understand and elucidate the roles (and possible interactions) of both humoral and cell-mediated immunity in the HPV-16/18 vaccine and the HPV-6/11/16/18 vaccine for protection against cervical lesions caused by oncogenic non-vaccine types. A limitation of our analysis is that it only evaluated the immune response to two oncogenic non-vaccine HPV types. The assessment of a greater number of non-vaccine types associated with cross-protection or a lack of cross-protection would be valuable to identify other immunological markers associated with cross-protection. This may increase our understanding of the underlying mechanism(s) of cross-protection, and with further follow-up, help to determine the duration of cross-protection and perhaps subsequently facilitate the determination of valid and universally accepted correlates of protection.