The data presented here demonstrate that a recombinant HPV-11 L1 VLP vaccine could elicit HPV-specific antibody and proliferative T-cell responses in 18- to 25-year-old women not exposed to HPV-11. All vaccine recipients had detectable proliferative responses following the second immunization, while vaccine-dependent antibody responses were observed immediately following the first priming immunization.
The results of this study do not indicate a correlation between the magnitude of antibody titer and proliferative T-cell responses in individual vaccine recipients. While it is thought that antibody production is facilitated by T-helper cells, the study results suggest that the dynamics of B- and T-cell populations within a single person cannot be analyzed in terms of a linear association between antigen-specific antibody secretion and lymphocyte proliferation. An increase in antibody titer was observed following each administration of the vaccine. This contrasted with lymphoproliferative responses that appear to achieve a maximum level of stimulation following initial immunization. Overall, HPV-specific T-cell activity was observed as a discrete proliferative response and there was no trend in average SI after the initial immunization. In other words, once the lymphoproliferative response was primed, the magnitude of that response did not change significantly with subsequent immunizations. This observation is consistent with the present understanding of the maintenance of homeostasis in memory T-cell populations (14
) and may be due to a “physiologic limit” of antigen-specific T cells that can be present in a host at any given time. Although there was no significant effect of vaccination on SI levels following the administration of the first immunization, it remains possible that the subsequent immunizations played a role in conferring long-lived proliferative T-cell responses.
To investigate whether HPV-11 VLP immunization could prime cross-reactive T-cell responses against other papillomavirus types, we performed lymphoproliferation assays on PBMCs from a subset of study participants and virginal women. Multiple papillomavirus VLP sources, including HPV-16 and BPV VLPs, were used for antigenic stimulation. A statistically significant increase between baseline proliferative responses and those measured 1 month following the third immunization was observed only against the HPV-11 VLP in vaccine recipients. Neither HPV-11 nor HPV-16 VLPs elicited lymphoproliferative responses in PBMCs isolated from virginal women. In both vaccinated and virginal women, BPV VLP most commonly elicited responses independent of the receipt of vaccine. In addition, mean responses against BPV VLP in vaccine participants were higher at baseline than after the receipt of three or four HPV-11 VLP immunizations, indicating that this response was not vaccine induced. Similarly, one of two HPV-16 VLP preparations (i.e., HPV-16 VLP produced in recombinant baculovirus-infected insect cells) elicited vaccine-independent responses in vaccine study participants at baseline, although the mean SI level generated was not above the cutoff for a positive SI value (Fig. ).
It is possible that some vaccine study participants had previous exposure to HPVs other than types 6 and 11 before their inclusion into this study; therefore, in vitro stimulation of PBMCs by BPV or HPV-16 VLPs may have induced a memory response against a conserved papillomavirus capsid epitope, albeit a putative common T-cell epitope has not been reported (20
). In fact, PCR-based HPV-16 DNA analysis of anogenital specimens from vaccine study participants over two years of the study revealed that two of the participants analyzed in these type-specific lymphoproliferation assays had been exposed to HPV-16 (data not shown). Both of these participants demonstrated HPV-16-specific lymphoproliferative memory responses and would therefore account for some of the HPV-16 type-specific proliferation observed.
The lymphoproliferative responses to BPV VLP by PBMCs from virginal women as well as from vaccine study participants pre-and post-HPV-11 VLP vaccination may be due to a general mitogenicity of the VLP structure, as has been suggested (30
). Other investigators have observed BPV VLP responses at baseline measurements in study participants of HPV-16 VLP-based vaccine trails (A. Hildesheim and L. Pinto, personal communication). Taken together, these data suggest that, whether due to a commonly recognized putative papillomavirus epitope or a general mitogenic stimulatory effect, use of BPV VLP as a “negative” VLP control appears inappropriate in VLP-based vaccine studies. Nonetheless, the robust increase in HPV-11 VLP lymphoproliferation observed after immunization with minimal vaccine-dependent responses to other VLPs supports the HPV type specificity of the measured proliferative response.
Our data on the type-specific nature of this VLP-based vaccine do not support recent findings by Evans and colleagues who reported cross-reactive cytokine and lymphoproliferative responses against HPV-6 and -16 following vaccination with HPV-11 VLP (13
). However, baseline measurements in vaccine recipients were high in the study by Evans et al. This may have been a consequence of previous exposure to other HPVs, given that study exclusion criteria were limited to a history of abnormal Pap smears and seropositivity to HPV-11 by ELISA only. HPV-6 and HPV-11 have 93% amino acid identity in the L1 protein yet do not share conformationally dependent neutralizing epitopes (7
); thus, it is not surprising that cross-type proliferative responses were not observed in the screening and ELISA evaluations. This may support future efforts to include both HPV-6 and HPV-11 in HPV VLP vaccines targeting low-risk HPV types.
Th1 and Th2 cytokines were detected in response to HPV-11 L1 VLP in vitro restimulation in all vaccine recipients and in none of the placebo recipients tested. In general, the quantitative responses of IFN-γ (picograms/milliliter) and IL-2 (per SIHT-2
) measured in vaccine recipients were similar to or exceeded previously reported levels of these cytokines detected after mitogenic (8
), E6/E7 peptide (53
), or VLP (11
) restimulation in women naturally infected with HPV-16. SIs against HPV-11 VLP were also higher than what is generally measured in naturally infected individuals (48
), suggesting that HPV VLP vaccination primes stronger cellular immune responses than natural HPV infection. Although IL-2 production was greater in the immunized group versus the placebo group following the second immunization, the differences were not statistically significant. It is possible that levels of IL-2 detected in the vaccine recipient group were low due to IL-2 uptake by VLP-stimulated PBMCs during 48 h of cell culture. While some significant associations between cytokine production, lymphoproliferation, and antibody titer were observed, these relationships were not consistently observed over time. This may be the result of the small number of participants evaluated in this study. Nonetheless, the observed correlations support the Th1/Th2 cytokine pattern measured among vaccinated participants.
In our study, immunoglobulin isotype and subclass responses to HPV-11 VLP vaccination demonstrated the generation of both Th1 and Th2 responses in over half of women immunized with HPV-11 VLP. These data corroborate a similar response observed in our cytokine and ELISPOT studies. Our results differ from those in the work previously published by Harro et al., which examined the immunoglobulin isotypes induced by vaccination with an HPV-16 VLP produced from baculovirus (19
). All vaccine recipients immunized with HPV-16 VLP (with or without aluminum adjuvant) were seropositive for HPV-16 VLP-specific IgG1 antibodies and minimal IgG2, IgG3, or IgG4 antibodies were detected 1 month following the first immunization. Similar frequencies of IgA were detected in women immunized with both HPV-11 and HPV-16 VLP vaccines, but IgM was more common in recipients of the HPV-16 vaccine, a likely consequence of the primary immune response generated immediately following vaccination. Several aspects of vaccine study design and the technologies employed may account for the differences in our results. We examined immune responses at month 7 after the vaccinees had received three doses of the HPV-11 VLP vaccine, whereas Harro et al. tested sera 1 month after the first vaccination. This may explain why we saw fewer individuals who had a detectable IgM response. This difference in the sampling time points may also explain why we were able to detect IgG2, IgG3, and IgG4 response in a higher percentage of individuals than that observed in the HPV-16 VLP vaccine study. The MAbs may have different affinities for the various immunoglobulin isotypes targeted and were obtained from different sources for the two studies. Lastly, our assay employed the use of Luminex microspheres and fluorescent detection technology as a readout, compared to a colorimetric detection used in an ELISA. The liquid phase kinetics of these microsphere-based binding assays, the increased sensitivity of fluorescence-based technology, and the increased precision afforded by analyzing multiple microspheres per well may account for the apparent increased sensitivity of the Luminex isotyping assay. Thus, the overall differences observed between our study and that reported by Harro et al. may be attributed to the laboratory technologies applied, the difference in HPV type-specific VLP selected for immunization, the vaccine formulation and doses administered, and the time point chosen for serologic measurement.
Although aluminum hydroxyphosphate is not known to be a potent cytotoxic-T-lymphocyte (CTL)-inducing adjuvant, immunization of VLPs without adjuvant has been shown to induce cell-mediated immune responses (10
). Recent observations of strong and long-lived CTL responses using a human immunodeficiency virus-gag
) and Th1 delayed-type hypersensitivity responses following HPV-6 VLP (55
), both in the absence of adjuvant, have been reported. In contrast, HPV-11 L1-specific CTLs were only weakly detectable by bulk CTL assays in our study after the fourth immunization in two of nine vaccine recipients monitored (data not shown). Our observations in humans corroborate the weak and infrequent CTLs measured in chimpanzees immunized with similar HPV VLP vaccines adsorbed to aluminum hydroxyphosphate (38
Detection of CTLs using techniques such as major histocompatibility complex tetramers and ELISPOT analysis have been reported to be more sensitive than bulk CTL assays (1
) and may provide a more accurate assessment of HPV-specific CD8+
-T-cell responses. Towards this aim we performed ELISPOT experiments on a subset of vaccine participants. The measurable IFN-γ cytokine response was generally restricted to the CD4+
population of T cells. It is possible that the addition of aluminum adjuvant to the HPV-11 VLP preparation interferes with the inherent ability of VLPs alone to prime a Th1-directed CTL response (10
Limited clinical investigations of human subjects with genital warts have revealed wart-infiltrating HPV-specific T cells to be modulators of disease outcome (2
). More extensive evidence exists that virus-specific cellular immune responses are protective against HPV-16 persistence and oncogenic development (3
), but the role of CD4+
activity in natural host protection remains uncertain. Although the generation of virus-specific cell-mediated immune responses appears to be beneficial for effective protection against chronic viral infections (33
), future work is needed to determine the respective role and relative contribution of HPV-specific host B and T cells in terms of protection against HPV-induced lesions. Similarly, the characterization of antibody, lymphoproliferative, and cytokine immune responses induced in patients by vaccination such as those presented here are potentially key to understanding the long-term efficacy of HPV VLP-based vaccines.