Robust In Vitro and In Vivo Neutralization against Multiple High-Risk HPV Types Induced by a Thermostable Thioredoxin-L2 Vaccine.

Author Manuscript Published OnlineFirst on July 13, 2015; DOI: 10.1158/1940-6207.CAPR-15-0164 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

HPV in vitro/in vivo neutralization induced by Trx-L2

Robust in vitro and in vivo neutralization against multiple highrisk HPV types induced by a thermostable thioredoxin-L2 vaccine

Hanna Seitz1*, Lis Ribeiro-Müller1, Elena Canali2, Angelo Bolchi2, Massimo Tommasino3, Simone Ottonello2,# Martin Müller1,#

1

German Cancer Research Center, Heidelberg, Germany

2

Department of Life Sciences, Biochemistry and Molecular Biology Unit, University of Parma, Italy 3

International Agency for Research on Cancer, Lyon, France

#

corresponding authors:

Martin Müller DKFZ ATV F035 Im Neuenheimer Feld 242 69120 Heidelberg, Germany [email protected] Tel. +49 6221 424628

Simone Ottonello Department of Life Sciences

Downloaded from cancerpreventionresearch.aacrjournals.org on August 12, 2015. © 2015 American Association for Cancer Research.


University of Parma Parco Area delle Scienze 23/A 43124 Parma, Italy [email protected] Tel. +39 521 905646

*present address: Laboratory of Cellular Oncology, NCI, 37 Convent Drive, Bethesda, MD 20814, USA

This work was supported by a grant from the Italian Association for Cancer Research to S. Ottonello (AIRC, grant IG 12956); E. Canali was supported by a post-doctoral fellowship from AIRC.

1

2 Downloaded from cancerpreventionresearch.aacrjournals.org on August 12, 2015. © 2015 American Association for Cancer Research.


Abstract Current prophylactic virus-like particle (VLP) human papillomavirus (HPV) vaccines are based on the L1 major capsid protein and provide robust but virus type-restricted protection. Moreover, VLP vaccines have a high production cost, require cold-chain storage and are thus not readily implementable in developing countries, which endure 85% of the cervical cancer death burden worldwide. In contrast to L1, immunization with minor capsid protein L2 elicits broad cross-neutralization and we previously showed that insertion of a peptide spanning amino acids 20-38 of L2 into bacterial thioredoxin (Trx) greatly enhances its immunogenicity. Building on this finding, we utilize, here, four different neutralization assays to demonstrate that low doses of a trivalent Trx-L2 vaccine, incorporating L2(20-38) epitopes from HPV16, HPV31 and HPV51, and formulated in a human-compatible adjuvant, induce broadly protective responses. Specifically, we show that this vaccine, which utilizes a far-divergent archaebacterial Trx as scaffold and is amenable to an easy one-step thermal purification, induces robust cross-neutralization against 12 of the 13 known oncogenic HPV types. Immune performance measured with two different in vitro neutralization assays was corroborated by the results of mouse cervico-vaginal challenge and passive transfer experiments indicating robust cross-protection also in vivo. Altogether our results attest to the potential of Trx-L2 as a thermostable second generation HPV vaccine particularly well suited for low-resource countries.



Introduction Infectious agents are responsible for approximately 2 million new cancer cases/year worldwide. Human papillomaviruses are responsible for 30% of infectioninduced cancers, most notably cervical cancer (1). In fact, after breast, colorectal and stomach cancer, cervical cancer is the fourth most frequent cancer in women. While cervical cancer is the most common HPV-induced malignancy in the developing world, HPV-related cancers including those of the anus and oropharynx are on the rise in developed countries, with a combined total incidence matching that of cervical cancer in the U.S. (2, 3). Eight years ago two prophylactic HPV vaccines, Gardasil and Cervarix, became available. Both are subunit vaccines composed of 360 monomers of the major capsid protein L1 assembled into non-infectious virus-like particles (VLPs). Gardasil, which is produced in yeast cells and adjuvanted with aluminum hydroxyphosphate sulphate, is tetravalent and contains VLPs targeting two oncogenic (HPV16 and HPV18) and two genital wart-causing (HPV6 and HPV11) viral types. Cervarix, which is produced in insect cells and formulated with a composite adjuvant (AS04, aluminum hydroxide plus monophosphoryl lipid A), is bivalent and only contains HPV16 and HPV18 VLPs. Since both vaccines include HPV16 and HPV18 VLPs, they provide ≥ 70% protection against cervical cancer. Standard vaccination includes three doses administered intramuscularly over 6 months, but a similar efficacy against HPV16 and HPV18 infections has been reported in women receiving three, two or even one dose(s) of Cervarix® (4). However, a likely disadvantage of fewer doses is reduction of the minimal cross-protection against



heterologous (“non-vaccine”) HPV types. Indeed, fewer doses of Cervarix® correlate with less HPV31 neutralization (5). In December 2014, a nonavalent vaccine targeting seven of the thirteen oncogenic HPVs, namely types 16, 18, 31, 33, 45, 52 and 58, was licensed by FDA. Besides inefficient cross-protection, another drawback of current VLP vaccines is their costly production/distribution platform which requires eukaryotic cell culture, laborious purification and cold-chain storage. In

contrast

to

conformation-dependent,

type-specific

L1-immunogens,

immunizations with linear, conserved regions of minor capsid protein L2 elicit broadly neutralizing responses. Cross-neutralizing epitopes have been mapped to various Nterminal regions of HPV16 L2 (e.g., amino acid residues 17-36, 20-38, 56-75 and 96-120) (6-8). Importantly, immunogens from different laboratories spanning amino acid (aa) residues 17-38 of L2, a region exposed on the cell surface during initial stages of infection, elicit cross-neutralization irrespective of the vaccine scaffold or formulation utilized. For example, concatenated aa 11-88 L2 fusion proteins elicit broad neutralization in mice and rabbits (9-11). Similarly, cross-neutralization was obtained with HPV16 and HPV31 17-36 L2 peptides displayed on adeno-associated virus (AAV) capsids (12). Also, chimeric HPV16-L1-VLPs bearing the 17-36 L2 peptide grafted onto the surface-exposed DE loop of L1 elicit durable and broadly neutralizing responses in mice and rabbits (13, 14). While production of chimeric HPV16 L1-L2 VLPs and AAV2L2(17-36)-HPV16-31 particles requires eukaryotic cell culture, bacteriophage-derived VLPs displaying 17-31 L2 yield a similarly broad protection even in the absence of adjuvant, and can be inexpensively purified from E. coli. (15-17).



We have successfully employed a non-virus display system based on bacterial thioredoxin as an immunogenicity-providing scaffold protein. Previously, we demonstrated that multi-peptide insertions of the 20-38 HPV16 L2 epitope into the active-site loop of E. coli thioredoxin creates an immunogen that elicits in vitrodetectable cross-neutralization in mice (18). These studies, however, were performed with high antigen doses (up to 100 µg) formulated in human-incompatible Freund’s adjuvant. Furthermore, cross-neutralization was not extended to notable high-risk (HR) HPV types including HPV31. We recently showed that a trivalent mixture of monovalent E. coli thioredoxin-L2 antigens bearing L2(20-38) epitopes from HPV16, HPV31 and HPV51 elicits a broader and more robust neutralization than individual monovalent antigens (19). We also showed that Trx-HPV16-L2 based on a far-divergent and hyperthermostable thioredoxin from Pyrococcus furiosus is as immunogenic as E. coli Trx-L2, but comes with the added benefits of (a) reduced cross-reactivity with human thioredoxin; (b) a simplified purification; and (c) a strikingly increased thermal stability (>12 h at 100°C, apparent Tm>95°C) (20). Taking into account the above scaffold improvement, the availability of different neutralization assays and the need for a L1-VLP comparison, we present here the results of a comprehensive immunization study evaluating the relative performance of monovalent P. furiosus thioredoxin-HPV16 L2(20-38)3 (PfTrx-16L2), a mixture of thioredoxin-L2 antigens derived from HPV16, 31, 51 (PfTrx-L2 mix), and HPV16-L1-VLPs. The PfTrx-L2 antigens were administered at moderate doses and formulated in the human-compatible adjuvant aluminum hydroxide-monophosphoryl lipid A (MPLA).



Neutralization was monitored in vitro using the L1- and the L2-pseudovirion-based neutralization assays (PBNA) and in vivo utilizing the cervico-vaginal challenge and passive transfer mouse model. Similarly protective responses for PfTrx-16L2 and PfTrxL2 mix, both broader than those attained with HPV16-L1-VLPs, were revealed by the challenge assay. However, passive transfer experiments and especially the L1-PBNA highlighted the more prominent cross-protection achieved with the PfTrx-L2 mix compared to PfTrx-16L2 and HPV16-L1-VLPs.



Materials and Methods

Expression and purification of the Trx-L2 proteins PfTrx-L2 constructs were generated by subcloning chemically synthesized sequences (Eurofins MWG Operon) coding for HPV16-, HPV31- and HPV51-L2(20-38)3 polypeptides into the CpoI site of an engineered version of Pyrococcus furiosus thioredoxin inserted into a 6xHis-tag-lacking pET-PfTrx plasmid. The resulting constructs were used for recombinant expression and one-step heat purification of the three Trx-L2 proteins as described previously (20). The composition and purity of protein preparations were assessed by electrophoretic analysis on 11-15% SDS-polyacrylamide gels and MALDI-TOF analysis. Protein concentration was determined by A280 measurements using calculated extinction coefficients as well as with the use of a Qubit® 2.0 Fluorometer (Life Technologies).

Expression and purification of HPV16-L1-VLPs Trichoplusia ni (TN) High Five cells (Invitrogen) were cultivated in Ex-CellTM 405 serumfree medium (SAFC Biosciences) at 27°C. Papillomavirus VLPs were produced as described previously (21). Purity and L1 content of individual fractions were assessed by SDS-PAGE and Coomassie-staining. Capsid quality was verified by electron microscopy.



Mouse and guinea pig and immunization Six- to eight-weeks-old female Balb/c mice were obtained from Charles River (Sulzfeld, Germany) and kept at the German Cancer Research Center under specific-pathogen-free conditions. Animals were immunized intramuscularly four times at monthly intervals with 15 µg PfTrx-L2 or HPV16-L1-VLPs adjuvanted with 50 µg aluminium hydroxide (Brenntag, Denmark) and 10 µg synthetic monophosphoryl lipid A (AvantiLipids, USA). Outbred Hartley (Crl:HA) guinea pigs (350-400 g female animals) were obtained from Charles River (Sulzfeld, Germany) and immunized intramuscularly four times at triweekly intervals with 50 µg PfTrx-L2 or HPV16-L1-VLPs formulated in 300 µg aluminum hydroxide and 20 µg synthetic monophosphoryl lipid A.

L1-pseudovirion-based neutralization assay HPV pseudovirions (PSV) were produced as described previously (22) with minor modifications (23). L1-PBNAs were performed as detailed recently (23) with a pseudovirion input of ∼0.5 ng L1 per 1 x 104 HeLaT cells, generated in house by stable large T-antigen transfection and routinely authenticated with the Multiplex human Cell line Authentication Test (MCA) (24).

L2 pseudovirion-based neutralization assay The L2-PBNA was performed as described (25) with a pseudovirion input of ∼2.5 ng L1 per 8 x 103 pgsa745 cells. All L2-PBNA cell lines, including pgsa745, CHOΔfurin and



MCF10A, were kindly provided by John Schiller’s lab (NIH, Bethesda MD) and were routinely authenticated with the MCA (24).

Challenge and passive transfer in the cervico-vaginal mouse model of HPV pseudovirion infection The mouse cervico-vaginal model was utilized as described (26) with minor modifications. Both challenge and passive transfer assays were performed over the course of eight days. On day 1, Balb/c male cage bedding was transferred to the cages of female mice to induce hormonal synchronization (Whitten effect). On day 3, 100 µl of 30 mg/ml Medroxyprogesteronacetat (Pharmacia, Ireland) was injected subcutaneously. In the case of passive transfer, 50 µl of serum (diluted 1:2 with PBS) were delivered intraperitoneally to each mouse on day 5. For challenge assays, no treatment was performed on day 5. On day 6, mice were treated with 50 µl of 4% Nonoxynol-9 (N9) (Spectrum, USA) plus 4% carboxymethylcellulose (CMC). Four hours after N9 treatment, HPV pseudovirions (∼250 ng L1/mouse for HPV16, 31, 33 and 51; ∼75 ng L1/mouse for HPV18) encapsidating a firefly luciferase plasmid were instilled intravaginally. Luminescence-based imaging using a Xenogen IVIS imager (Xenogen Corporation-Perkin Elmer, USA) was performed on day 8. Images were acquired before and after intravaginal instillation of 20 µl luciferin substrate (15 mg/ml; Promega, USA). Three minutes after substrate addition, luminescence was recorded (30 seconds–1 minute exposure time, medium binning). A region-of-interest (ROI) analysis was performed using the Living Image 2.50.1 software (Xenogen-Perkin Elmer, USA).



Statistical analysis Statistical analysis was performed with GraphPad Prism 5.00 (GraphPad Software, USA) using the non-parametric Mann-Whitney test; differences were considered significant at p ≤ 0.05.



Results To move forward with thioredoxin-L2 as a second generation HPV vaccine, we initially focused on antigenic dose and adjuvant formulation using monovalent Trx-16L2 as a test antigen. We found that Trx-16L2 doses comprised between 50 ng and 15 µg elicit potent type-specific and cross-neutralizing responses (Figure 1). We tested several humancompatible adjuvants and selected aluminum hydroxide-MPLA because it produced neutralization titers most comparable to those achieved with Freund’s adjuvant (data not shown). The lead thioredoxin-L2 vaccine investigated in this work is an equimolar mixture of three monomeric, thermally purified and fully oxidized Pyrococcus furiosus (Pf) Trx-L2 proteins (“mix”), each bearing a three-fold repeated L2(20-38) peptide from HPV16, 31 or 51. The PfTrx-L2 mix was compared to monovalent PfTrx-16L2 and HPV16-L1-VLPs, and neutralizing responses were analyzed in vitro on groups of 35 Balb/c mice per immunogen using the standard L1-PBNA and the L2-PBNA (25). Twenty-five of the 35 mice in each group were randomly selected and destined to in vivo vaginal challenge with pseudovirions from five different oncogenic HPV types. Final sera were collected from the remaining 10 animals in each group, including 10 negative control animals immunized with the empty PfTrx scaffold, pooled, L1-PBNA titrated and passively transferred into naïve mice.



Superior in vitro cross-neutralization capacity of the PfTrx-L2 mix formulation Intermediate sera were collected from mice vaccinated with PfTrx-L2 mix, PfTrx-16L2 or HPV16-L1-VLPs two weeks after the final immunization and analyzed on HPV16, 18, 31, 33 and 51 pseudovirions in the L1- and L2-PBNAs (Figure 2). Consistent with our previous results (19), the PfTrx-L2 mix elicited comparable HPV16 type-specific responses (Figure 2A) in addition to significantly broader cross-reactivity (Figure 2B-2E). Its robustness and cross-neutralization capacity were especially apparent in the L2PBNA, where most sera neutralized HPV31 and HPV51 pseudovirions well above the 50% threshold (Figure 2B/2C). Also in line with previous findings, monovalent PfTrx16L2 did not elicit cross-neutralizing responses against HPV31 and HPV51, but effectively neutralized HPV18 and HPV33 (Figure 2D/2E). While HPV16-L1- VLPs induced exceptionally strong HPV16 neutralization responses, they largely failed at crossneutralization. In particular, anti-HPV16-L1-VLP immune sera showed neither HPV18 nor HPV51 cross-neutralization capacity and limited activity against HPV31 and HPV33. These results are consistent with the observation that Cervarix, which also contains aluminum hydroxide-MPLA as adjuvant, is able to induce cross-protection against HPV31 and HPV33 (27). To determine in vitro cross-neutralization titers, pools of the 35 sera were titrated against 13 HPV pseudovirion types presently recognized as “high-risk” (HPV16, 18, 45, 31, 33, 52, 58, 35, 59, 56, 51, 39 and 68) (28). Three different pools from PfTrx16L2 and PfTrx-L2 mix immune sera were generated based on the magnitude of their HPV16 neutralization titers. Pool #1 included sera with HPV16 titers greater than 2,000,



while pool #2 contained sera with titers greater than 1,000 but lower than 2,000, and pool #3 comprised sera with titers below 1,000. For HPV16-L1-VLP immune sera, two pools were created: pool #1 comprised of sera with anti-HPV16 titers greater than 3,000,000 and pool #2 with titers of 3,000,000 or lower. The PfTrx-L2 mix sera, especially the high-titer pool #1, performed best in terms of neutralization breadth and potency, with neutralization activity against all 13 pseudovirions except HPV56 (Table 1). The PfTrx-16 L2 sera, specifically the high- and medium-titer pools (#1 and #2), neutralized 10 of the 13 pseudovirions, with no activity against HPV31, 51, 59 and 56. As expected, HPV16-L1-VLP sera showed high type-specific neutralization against HPV16, but minimal cross-neutralization against two (pool #1) or four (pool #2) of the other tested pseudovirions. Importantly, and in stark contrast with the outstanding titers measured against HPV16, the cross-neutralization capacity of anti-VLP immune sera was particularly low on HPV31, 33, 58, 35 and HPV56 pseudovirions. Notably, anti-HPV16-L1VLP pool #1 and pool #2 displayed significantly different cross-neutralization patterns. While pool#1 cross-neutralized HPV58 and HPV56, pool #2 cross-neutralized HPV31, 33, 35 and 56.

PfTrx-L2 mix and PfTrx-16L2 immunized mice are similarly protected from vaginal in vivo challenge with heterologous HPV types Twenty-five mice each from the PfTrx-L2 and L1-VLP groups were challenged with HPV16, 31, 51, 33 or 18 pseudovirions (5 mice per HPV type). The rationale for choosing these particular HR types rests on the fact that HPV16 and HPV18 are the two most



prominent HPV types associated with cervical cancer, while HPV31, HPV33 and HPV51 are the most divergent (from HPV16) within the aa. 17-38 epitope region of L2 and thus often escape HPV16 L2 antigen-induced neutralization. Luciferase activity and hence the degree of infection was measured two days after pseudovirion instillation, (Figure 3). In vivo measured luminescence signals are reported in Figure 3A-3E; a representative image of the vaginal in vivo challenge with HPV51 is shown in Figure 3F. As expected, mice immunized with PfTrx-16L2, PfTrx-L2 mix or HPV16-L1-VLPs were all protected from HPV16 challenge (Figure 3A). Significant protection was also observed for HPV31, with the mix performing best (Figure 3B). Interestingly, although HPV31 neutralization could not be measured in vitro in most sera from PfTrx-16L2 and HPV16-L1-VLP immunized animals before challenge, mice in these groups were protected from in vivo HPV31 infection. PfTrx-L2 mix immunized animals, three of which displayed L1-PBNA-detectable HPV51 titers, were also significantly protected from HPV51 pseudovirion challenge (Figure 3C, 3F). Measurable HPV51 in vitro neutralization titers in four of the five mice immunized with PfTrx-16L2 were corroborated by in vivo protection, although significance could not be determined due to a high infection rate in one animal. Curiously, HPV16-L1-VLP immunized mice seemed more susceptible to HPV51 infection than negative control animals. HPV18 challenge results proved difficult to interpret due to the unusually low transduction efficiency obtained with this particular HPV type, with eight out 10 negative control animals not infected (Figure 3D). Nevertheless, PfTrx-16L2 and PfTrx-L2 mix immunized mice appeared to be protected and all yielded significant L1-PBNA titers, with no or very little in vivo detectable firefly



luciferase activity. In contrast, L1-VLP-immunized mice were infected and to a greater extent than negative control animals. PfTrx-16L2 and PfTrx-L2 mix immunized animals were similarly protected from challenge with HPV33, with higher L1-PBNA titers for the latter antigen formulation (Figure 3E). By comparison, no in vitro titers nor protection against HPV33 challenge were observed in L1-VLP-immunized mice (with the exception of one animal).

Passive transfer of PfTrx-L2 mix immune sera provides more robust cross-protection than PfTrx-16L2 or L1-VLP immune sera As revealed by the above data the challenge assay is extremely sensitive and capable to uncover neutralization activities undetectable in vitro. Multiple immune mechanisms, including innate and cellular immunity in addition to humoral immunity, likely contribute to protection in this experimental set-up. Instead, passive transfer of immune-sera is humorally defined and probably only relies on vaccine-induced antibodies. After the final immunization, sera from ten mice in each group (PfTrx-16L2, PfTrx-L2 mix, HPV16-L1-VLP and empty PfTrx negative control) were pooled and L1PBNA titrated against HPV16, HPV31, HPV51, HPV18 and HPV33 pseudovirions. Pools were passively transferred intraperitoneally into five groups of animals, which were subsequently infected with the above pseudovirions (Figure 4). As observed in the challenge and anticipated by the high L1-PBNA titers, passively transferred sera (diluted ~1:40; serum:blood volume ratio) from PfTrx-16L2-, PfTrx-L2 mix- and HPV16-L1-VLP-immunized mice provided protection against HPV16



infection (Figure 4A). However, at variance with the challenge results, where PfTrx-16L2 and HPV16-L1-VLPs immunized mice were protected from HPV31 regardless of their in vitro titers (cf. Figures 2B and 3B), passively transferred PfTrx-16L2 and HPV16-L1-VLP sera without a detectable HPV31 L1-PBNA titer failed to confer protection against HPV31 (Figure 4B). In fact, only the PfTrx-L2 mix serum pool with an L1-PBNA titer of 300 provided HPV31 protection upon passive transfer. Instead, both PfTrx-16L2 and PfTrx-L2 mix sera afforded protection against HPV51, HPV18 and HPV33 (Figure 4C-E). Importantly, however, protection conferred by the PfTrx-L2 mix sera was significantly more robust. Under the same conditions, passively transferred HPV16-L1-VLP sera lacking L1-PBNA measurable titers against these three HPV types did not provide any protection against HPV51, HPV18 or HPV33 (Figure 4C-E).

PfTrx-L2 immunogens elicit higher magnitude responses in guinea pigs than in mice Despite the favorable results obtained in Balb/c mice, inbred animals only partially mirror the human response and parallel studies on additional animal models are desirable, and required, for further vaccine development. To this end, we immunized outbred guinea pigs with a subset of PfTrx-L2 immunogens including PfTrx-16L2, PfTrx31L2, PfTrx-51L2, the PfTrx-L2 mix, and HPV16-L1-VLPs, adjuvanted with alum-MPLA, using the same protocol applied to Balb/c mice. Final sera were titrated in the L1-PBNA against HPV16, HPV31, HPV51, HPV18, HPV45, HPV33, HPV59 and HPV56 pseudovirions (Table 2).



When comparing PfTrx-L2-induced immune responses in mice and guinea pigs, it is apparent that HPV16 and HPV51 neutralization is higher in the latter species. In contrast, L1-VLPs induced equally high HPV16 titers in both species. Interestingly, and in contrast with what we observed in mice (Table 1), PfTrx-16L2 elicited similar HPV51 neutralization titers as PfTrx-51L2 in guinea pigs. Also at variance with the situation in mice, PfTrx-16L2 elicited weak HPV18 neutralization responses in guinea pigs. However, PfTrx-L2 mix and PfTrx-51L2, both induced strong HPV18 neutralization responses in guinea pigs. In this species, PfTrx-L2 mix yielded rather low HPV31 neutralization, whereas the opposite was observed for HPV16-L1-VLPs. Thus, despite the rather small sample size, cross-neutralization in guinea pigs seems to be somewhat different from that in mice. However, the best immune performance was still achieved with the PfTrxL2 mix.



Discussion We recently demonstrated the superior cross-neutralization responses elicited by a trivalent mixture of E. coli thioredoxin-L2 immunogens bearing L2 sequences from HPV16, 31 and 51 compared to the corresponding monovalent antigens (19). Since the latter study only measured in vitro neutralization, we wished to further investigate the immune performance of the Trx-L2 mix via a comprehensive set of in vivo (vaginal challenge and passive transfer) and in vitro (L1- and L2-PBNA) assays, using monovalent Trx-16L2 and HPV16-L1-VLPs as reference immunogens. In doing so, we also gained information on the relative sensitivity and predictive value of the four assays. Another major variation introduced in the present study was the use of heat-purified PfTrx-L2 immunogens built on the non-cross-reactive and highly thermostable thioredoxin from archaebacterium Pyrococcus furiosus (Pf) (20). L1- and L2-PBNA data corroborated our previous finding that the trivalent PfTrx-L2 mix elicits stronger neutralization against HPV31 and HPV51 compared to PfTrx-16L2 and HPV16-L1-VLPs. Moreover, PfTrx-L2 mix induced higher-level neutralization of the nonvaccine type HPV33 and neutralization of HPV18 similar to that of PfTrx-16L2. L1-PBNA titrations of pooled sera against the 13 oncogenic HPV types revealed the superior crossneutralization capacity of sera from mice immunized with PfTrx-L2 mix, which neutralized 12 of the 13 tested oncogenic HPV types (Table 1). By comparison, sera from mice immunized with PfTrx-16L2 or HPV16-L1-VLPs neutralized ten and five oncogenic HPV types, respectively. Interestingly, in vivo challenge experiments revealed protective effects in animals immunized with PfTrx-L2 mix, monovalent PfTrx-16L2 and HPV16-L1-VLPs, even in the absence of measurable in vitro titers. This was especially evident in animals immunized



with PfTrx-16L2 and subsequently challenged with HPV31, HPV51 or HPV33 pseudovirions. In fact, if one only considered challenge data, PfTrx-16L2 would appear comparable to the PfTrx-L2 mix with regard to cross-protection breadth. Passive transfer data, instead, were more aligned with those obtained from in vitro neutralization assays. Only sera with a measurable in vitro neutralization titer conferred protection in vivo upon transfer to naïve mice. Also, higher-titer sera generally afforded greater in vivo protection than lower-titer sera. Thus, based on in vitro neutralization and in vivo passive transfer data, the PfTrx-L2 mix appears to induce the broadest cross-neutralization, while an immunogenicity similar to that of PfTrx-16L2 is supported by challenge data. If we rank the four assays according to their sensitivities, the in vivo challenge and the L1-PBNA emerge as the most and the least sensitive, respectively, while the L2-PBNA and the passive transfer (with a serum transfer volume of 50 μl) are of intermediate sensitivity. How long-lived can be a neutralization that cannot be measured in vitro? What immune mechanisms promote the apparently extended protection observed in the challenge assay? Which assay represents the most reliable correlate of multi-type HPV protection in humans? Neutralization measured in vitro reflects prevention of virus internalization by anticapsid antibodies. However, additional mechanisms likely enhance antibody-mediated neutralization, including complement binding, FcR engagement and recruitment of phagocytic immune cells. These mechanisms are not recapitulated in vitro but probably contribute to neutralization efficiency measured in the challenge assay. Moreover, innate components such as complement, IgM, macrophages and neutrophils, might play a significant role in in vivo neutralization. Although these in vivo-restricted mechanisms



undoubtedly contribute to the overall response, an ideal L2 immunogen should elicit in vitro and in vivo neutralization. While in vitro assays may underestimate in vivo neutralization capacity, they provide a reliable, albeit indirect indication on immunity robustness. Hence, it is reassuring that the PfTrx-L2 mix elicits L1-PBNA detectable titers against 12 of the 13 oncogenic HPV types. The latter results as well as those produced by the other assays were obtained at a fixed time (2 months) after the last vaccination. Thus, longevity of immunization-induced anti-HPV protection still needs to be addressed in future studies. Interestingly, PfTrx-L2 immunogens elicited higher HPV16 and HPV51 neutralization titers in guinea pigs than in mice, while L1-VLPs induced similarly high HPV16 neutralizing responses in both species. Furthermore, and in contrast with the situation in mice (19), PfTrx16L2 extended neutralization to HPV51 in guinea pigs as effectively as PfTrx-51L2. However, fairly low anti-HPV31 neutralization titers were induced in guinea pigs by PfTrx-L2 mix, while the opposite was observed with HPV16-L1-VLPs. In summary, slightly different immune responses, in terms of magnitude and breadth, were observed with PfTrx-L2 and HPV16-L1VLPs in guinea pigs compared to mice. In both animals, however, PfTrx-L2 mix was the most effective immunogen. Why does PfTrx-L2 elicit higher neutralization in guinea pigs than in mice? Conceivably, immunoglobulin germ-line configurations may be more adapted to recognize, and be activated by, PfTrx-L2. Also, the TLR-4 agonist monophosphoryl lipid A may be more effective in guinea pigs than in mice (29). Also, while both Balb/c mice and guinea pigs express TLR-4 receptors on dendritic cells, signaling might be more sustained in the latter species. Although PfTrx-L2-associated T-helper epitopes have not been mapped yet,



preliminary data indicate that such epitopes can have a strong, strain-dependent impact on immunogenicity and the immunogenicity plunge observed in certain mouse strains can be compensated by incorporation of pan-reactive T-helper epitopes. Ultimately, it is encouraging to see that even unmodified PfTrx-L2 immunogens elicit cross-neutralizing responses in more than one preclinical model organism. Despite recent progress in the optimization of L2-based immunogens (11, 13, 30) the current benchmark of HPV protection is set by the licensed VLP vaccines. HPV16 neutralization endpoints measured with the HPV16-L1-VLPs we used as a surrogate L1vaccine reference were nearly identical to those achieved with PfTrx-L2, both the monovalent and the mix formulation. However, similar to the results obtained in other L1 vs. L2 vaccine comparisons (10, 14), neutralization titers were two-three orders of magnitude higher for L1-VLPs than for PfTrx-L2. This gap, whose clinical significance is unknown, was largely compensated by the superior cross-neutralization achieved with the PfTrx-L2 mix. Cross-protection remains the top goal of current VLP-HPV vaccine development. This is well documented by the recent FDA licensing of a nonavalent variant of Gardasil (V503), which includes five additional high-risk VLP types (HPV31, 33, 45, 52, and 58) besides HPV16, 18, 6 and 11. Despite the extended coverage of V503, its breadth of effective cross-protection remains to be determined. In addition to the six oncogenic types not present in V503, this includes a number of (a) alpha-types presently considered as low-risk and (b) non-genital types which may be (co)causal agents of non-melanoma skin cancer, especially in immunocompromised patients. Given the type-specificity of L1, it is unlikely that the nonavalent vaccine will confer any appreciable cross-protection against these HPV types,



while we know, for example, that the PfTrx-L2 vaccine induces significant L1-PBNA titers against the dermatotropic alpha type HPV5 implicated in squamous cell carcinomas (Martin Müller, unpublished results). The increased complexity of the nonavalent vaccine may increase production costs and further reduce physicochemical (especially thermal) stability. This, in turn, makes the vaccine even less accessible to the populations most in need of HPV protection, i.e., low resource countries which bear 85% of the global cervical cancer burden, often more strongly associated with types other than HPV16 and HPV18 (31). Along this view, the PfTrx-L2 vaccine embodies relevant features of a next generation HPV vaccine, namely strong HPV16 and HPV18 protection coupled to extended cross-protection, thermal stability and cost-effectiveness.



References 1. de Martel C, Ferlay J, Franceschi S, Vignat J, Bray F, Forman D, et al. Global burden of cancers attributable to infections in 2008: a review and synthetic analysis. The lancet oncology. 2012;13:607-15. 2. Chaturvedi AK, Anderson WF, Lortet-Tieulent J, Curado MP, Ferlay J, Franceschi S, et al. Worldwide trends in incidence rates for oral cavity and oropharyngeal cancers. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2013;31:45503. Chaturvedi AK, Engels EA, Pfeiffer RM, Hernandez BY, Xiao W, Kim E, et al. Human papillomavirus and rising oropharyngeal cancer incidence in the United States. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2011;29:4294301. 4. Kreimer AR, Rodriguez AC, Hildesheim A, Herrero R, Porras C, Schiffman M, et al. Proof-of-principle evaluation of the efficacy of fewer than three doses of a bivalent HPV16/18 vaccine. Journal of the National Cancer Institute. 2011;103:1444-51. 5. Safaeian M, Porras C, Pan Y, Kreimer A, Schiller JT, Gonzalez P, et al. Durable antibody responses following one dose of the bivalent human papillomavirus L1 virus-like particle vaccine in the Costa Rica Vaccine Trial. Cancer Prev Res (Phila). 2013;6:1242-50. 6. Gambhira R, Karanam B, Jagu S, Roberts JN, Buck CB, Bossis I, et al. A protective and broadly cross-neutralizing epitope of human papillomavirus L2. Journal of virology. 2007;81:13927-31. 7. Kondo K, Ishii Y, Ochi H, Matsumoto T, Yoshikawa H, Kanda T. Neutralization of HPV16, 18, 31, and 58 pseudovirions with antisera induced by immunizing rabbits with synthetic peptides representing segments of the HPV16 minor capsid protein L2 surface region. Virology. 2007;358:266-72. 8. Nakao S, Mori S, Kondo K, Matsumoto K, Yoshikawa H, Kanda T. Monoclonal antibodies recognizing cross-neutralization epitopes in human papillomavirus 16 minor capsid protein L2. Virology. 2012;434:110-7. 9. Jagu S, Karanam B, Gambhira R, Chivukula SV, Chaganti RJ, Lowy DR, et al. Concatenated multitype L2 fusion proteins as candidate prophylactic pan-human papillomavirus vaccines. Journal of the National Cancer Institute. 2009;101:782-92. 10. Jagu S, Kwak K, Karanam B, Huh WK, Damotharan V, Chivukula SV, et al. Optimization of multimeric human papillomavirus L2 vaccines. PLoS One. 2013;8:e55538. 11. Jagu S, Kwak K, Schiller JT, Lowy DR, Kleanthous H, Kalnin K, et al. Phylogenetic considerations in designing a broadly protective multimeric L2 vaccine. Journal of virology. 2013;87:6127-36. 12. Nieto K, Weghofer M, Sehr P, Ritter M, Sedlmeier S, Karanam B, et al. Development of AAVLP(HPV16/31L2) particles as broadly protective HPV vaccine candidate. PLoS One. 2012;7:e39741. 13. Schellenbacher C, Kwak K, Fink D, Shafti-Keramat S, Huber B, Jindra C, et al. Efficacy of RG1-VLP Vaccination against Infections with Genital and Cutaneous Human Papillomaviruses. The Journal of investigative dermatology. 2013.



14. Schellenbacher C, Roden R, Kirnbauer R. Chimeric L1-L2 virus-like particles as potential broad-spectrum human papillomavirus vaccines. Journal of virology. 2009;83:10085-95. 15. Tumban E, Peabody J, Peabody DS, Chackerian B. A pan-HPV vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing epitopes from the HPV minor capsid protein, L2. PLoS One. 2011;6:e23310. 16. Tumban E, Peabody J, Peabody DS, Chackerian B. A universal virus-like particle-based vaccine for human papillomavirus: longevity of protection and role of endogenous and exogenous adjuvants. Vaccine. 2013;31:4647-54. 17. Tumban E, Peabody J, Tyler M, Peabody DS, Chackerian B. VLPs displaying a single L2 epitope induce broadly cross-neutralizing antibodies against human papillomavirus. PLoS One. 2012;7:e49751. 18. Rubio I, Bolchi A, Moretto N, Canali E, Gissmann L, Tommasino M, et al. Potent antiHPV immune responses induced by tandem repeats of the HPV16 L2 (20 -- 38) peptide displayed on bacterial thioredoxin. Vaccine. 2009;27:1949-56. 19. Seitz H, Canali E, Ribeiro-Muller L, Palfi A, Bolchi A, Tommasino M, et al. A three component mix of thioredoxin-L2 antigens elicits broadly neutralizing responses against oncogenic human papillomaviruses. Vaccine. 2014;32:2610-7. 20. Canali E, Bolchi A, Spagnoli G, Seitz H, Rubio I, Pertinhez TA, et al. A high-performance thioredoxin-based scaffold for peptide immunogen construction: proof-of-concept testing with a human papillomavirus epitope. Scientific reports. 2014;4:4729. 21. Muller M, Zhou J, Reed TD, Rittmuller C, Burger A, Gabelsberger J, et al. Chimeric papillomavirus-like particles. Virology. 1997;234:93-111. 22. Buck CB, Thompson CD. Production of papillomavirus-based gene transfer vectors. Current protocols in cell biology / editorial board, Juan S Bonifacino [et al]. 2007;Chapter 26:Unit 26 1. 23. Seitz H, Dantheny T, Burkart F, Ottonello S, Muller M. Influence of oxidation and multimerization on the immunogenicity of a thioredoxin-l2 prophylactic papillomavirus vaccine. Clinical and vaccine immunology : CVI. 2013;20:1061-9. 24. Castro F, Dirks WG, Fahnrich S, Hotz-Wagenblatt A, Pawlita M, Schmitt M. Highthroughput SNP-based authentication of human cell lines. Int J Cancer. 2013;132:308-14. 25. Day PM, Pang YY, Kines RC, Thompson CD, Lowy DR, Schiller JT. A human papillomavirus (HPV) in vitro neutralization assay that recapitulates the in vitro process of infection provides a sensitive measure of HPV L2 infection-inhibiting antibodies. Clinical and vaccine immunology : CVI. 2012;19:1075-82. 26. Roberts JN, Buck CB, Thompson CD, Kines R, Bernardo M, Choyke PL, et al. Genital transmission of HPV in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan. Nature medicine. 2007;13:857-61. 27. Draper E, Bissett SL, Howell-Jones R, Edwards D, Munslow G, Soldan K, et al. Neutralization of non-vaccine human papillomavirus pseudoviruses from the A7 and A9 species groups by bivalent HPV vaccine sera. Vaccine. 2011;29:8585-90. 28. Bouvard V, Baan R, Straif K, Grosse Y, Secretan B, El Ghissassi F, et al. A review of human carcinogens--Part B: biological agents. The lancet oncology. 2009;10:321-2.



29. Cambier L, Bagut ET, Heinen MP, Tabart J, Antoine N, Mignon B. Assessment of immunogenicity and protective efficacy of Microsporum canis secreted components coupled to monophosphoryl lipid-A adjuvant in a vaccine study using guinea pigs. Veterinary microbiology. 2015;175:304-11. 30. Tyler M, Tumban E, Dziduszko A, Ozbun MA, Peabody DS, Chackerian B. Immunization with a consensus epitope from human papillomavirus L2 induces antibodies that are broadly neutralizing. Vaccine. 2014;32:4267-74. 31. Tjalma WA. There are two prophylactic human papillomavirus vaccines against cancer, and they are different. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2015;33:964-5.



Tables PfTrx-16L2 #1

HPV16 HPV18 HPV45 HPV31 HPV33 HPV52 HPV58 HPV35 HPV59 HPV56 HPV51 HPV39 HPV68b

PfTrx-L2 mix

HPV16-L1-VLPs

#2

#3

#1

#2

#3

#1

#2

6,171

2,701

718

7,020

1,922

590

8,306,000

1,869,000

671

494

255

1,179

339

151

-

-

380

357

215

987

397

194

-

-

-

-

-

250

273

104

-

146

210

253

-

574

199

111

-

105

184

274

-

593

189

-

-

-

1,580

2,086

437

5,298

1,972

683

393

-

197

190

-

516

204

-

-

648

-

-

-

282

218

165

-

-

-

-

-

-

-

-

128

119

-

-

-

159

-

-

111

135

-

325

117

-

-

-

278 168 400 149 Table 1. L1-PBNA titers (IC50 values) of serum pools #1, #2 and #3 derived from PfTrx-16L2, PfTrx-L2 mix or HPV16-L1-VLPs vaccinated mice. Sera from 35 animals immunized with the indicated antigens were collected two weeks after the fourth immunization and titrated against the 13 high-risk pseudovirions. For PfTrx-16L2 and PfTrx-L2 mix, pool #1 included sera with HPV16 neutralization titers higher than 2,000; pool #2 comprised sera with titers greater than 1,000; and pool #3 contained sera with neutralization titers below 1,000. For HPV16-L1-VLPs, pool #1 comprised sera with anti-HPV16 neutralization titers higher than 3,000,000, whereas pool #2 contained sera with titers below 3,000,000.

PfTrx-16L2 PfTrx-31L2 PfTrx-51L2 PfTrx-L2 mix HPV16-L1-VLPs #1 #2 #1 #2 #1 #2 #1 #2 #1 #2 HPV16 41,115 120,696 14,982 4,793 190,492 8,037 34,450 8.5x106 7.1x106 HPV31 184 2,300 73 7,485 7,228 HPV51 1,247 3,537 465 267 4,826 294 2,650 HPV18 115 230 604 1,474 1,124 901 1,537 HPV45 428 443 943 926 721 257 597 HPV33 981 232 HPV59 118 1,090 88 HPV56 Table 2. L1-PBNA titers (IC50 values) of immune sera from PfTrx-16L2, PfTrx-31L2, PfTrx-51L2, PfTrx-L2 mix or HPV16-L1-VLPs immunized guinea pigs. Guinea pigs (2 animals/group) were immunized intramuscularly four times at triweekly intervals with 50 µg antigen formulated in Alum-MPLA. Final sera were collected eight weeks after the fourth immunization and titrated in the L1-PBNA.



Figure Legends Figure 1. Dose-effect analysis of the monovalent Pyrococcus furiosus (Pf) Trx-16L2 antigen. Mice were immunized intramuscularly four times at monthly intervals with the PfTrx-16L2 antigen formulated in Alum-MPLA. Intermediate sera were collected two weeks after the third immunization and L1-PBNA titrated against HPV16 (left) and HPV18 (right) pseudovirions; dots represent neutralization titers against the indicated HPV types measured in individual mouse sera. Antigen doses between 1 µg and 15 µg elicit higher type-specific and cross-neutralizing titers than 50 ng-200 ng doses. Mean titers are indicated by horizontal bars; statistical significance of the differences between the immune responses elicited by different antigen doses was determined with the Mann-Whitney test; ns = non-significant.

Figure 2. Cross-neutralization broadness: comparison between monovalent PfTrx-16L2, PfTrx-L2 mix and HPV16-L1-VLPs. Sera collected two weeks after the fourth immunization were tested at a 1:200 dilution against HPV16 (A), HPV31 (B), HPV51 (C), HPV18 (D) and HPV33 (E) pseudovirions using the standard L1- and the modified L2-PBNAs (left and right columns, respectively). Each dot represents one mouse serum; data are the mean plus standard deviation (SD) of neutralization percentages measured within individual assay groups.

Figure 3. Broader in vivo challenge protective responses elicited by monovalent and mix PfTrx-L2 formulations compared to HPV16-L1-VLPs. After four immunizations with the indicated immunogens, sera from five animals were collected and analyzed in the L1-PBNA (individual titers are reported below each immunogen). Immunized mice were subsequently challenged with HPV16 (A), HPV31 (B), HPV51 (C), HPV18 (D) and HPV33 (E) pseudovirions. A similarly broad protection against the five tested HPV types was detected with sera from PfTrx-L2-vaccinated



mice, despite low (or absent) L1-PBNA titers in some animals. A type-restricted (HPV16 and HPV31 only) protection is apparent, instead, in HPV16-L1-VLP-immunized animals. Average radiance values indicating the extent of infection are reported for each mouse; non-immunized animals served as negative controls. Representative images showing the magnitude of vaginal infection by HPV51 PSVs are shown in 2F. The colors (scale shown on the left) indicate the intensity of luciferase expression; a region-of-interest (ROI) analysis for average radiance (p/s/cm2/sr) was performed with the Living Image 2.50.1 software.

Figure 4.

Broader protection afforded by anti-PfTrx-L2 mix sera compared to sera from

monovalent PfTrx-16L2-immunized animals. After four immunizations with the empty PfTrx scaffold, PfTrx-16L2, PfTrx-L2 mix and HPV16-L1-VLPs, sera from 10 mice were collected, pooled, L1-PBNA titrated and passively transferred into naïve mice. Recipient animals were then infected with HPV16 (A), HPV31 (B), HPV51 (C), HPV18 (D) or HPV33 (E) pseudovirions. The broader protection afforded by mix-elicited antibodies compared to monovalent PfTrx-L2 is apparent in panels B, C and E; a significantly lower cross-protection against all viral types except HPV16 was observed with anti-HPV16-L1-VLP sera. Data are average radiance values, indicating the extent of infection in individual mice. L1-PBNA titers of individual serum pools are shown on a gray background above each group; the lack of a value, as in the case of PfTrx-16L2 in panel B, indicates the absence of a measurable neutralization titer.


Figure 1

HPV18 Neutralization Titer

µg 15

µg

µg


5

15

5

1

0 20

50

1

10

ng

10

ns

ns

p = 0.0051

0

100

µg

100

µg

1,000

µg

1,000

ng

10,000

ng

10,000

ns

ng

p = 0.0482 p = 0.0145

100,000

ns

ns

50

100,000

20

HPV16 Neutralization Titer

Figure 2 (A) p < 0.0001

p < 0.0001

p < 0.0001

100

% Neutralization

% Neutralization

HPV16 L2 PBNA

HPV16 L1 PBNA

80 60 40 20

100 80 60 40 20 0

0

PfTrx-16L2

(B)

PfTrx-L2 mix

PfTrx-16L2

HPV16 L1 VLPs

PfTrx-L2 mix

HPV16 L1 VLPs

HPV31 L2 PBNA

HPV31 L1 PBNA p < 0.0001

p < 0.0001

100

p = 0.0001

% Neutralization

% Neutralization

p < 0.0001

80 60 40 20

p < 0.0001

100 80 60 40 20 0

0

PfTrx-16L2

PfTrx-L2 mix

HPV16 L1 VLPs

PfTrx-16L2

PfTrx-L2 mix


HPV16 L1 VLPs

Figure 2 (C)

p < 0.0001

p = 0.0005

p < 0.0001

100

% Neutralization

% Neutralization

HPV51 L2 PBNA

HPV51 L1 PBNA

80 60 40 20 0

100 80 60 40 20 0

PfTrx-16L2

PfTrx-L2 mix

PfTrx-16L2

HPV16 L1 VLPs

(D)

PfTrx-L2 mix

p < 0.0001

p < 0.0001

% Neutralization

100 80 60 40 20 0

PfTrx-16L2

PfTrx-L2 mix

HPV16 L1 VLPs

HPV18 L2 PBNA

HPV18 L1 PBNA

% Neutralization

p < 0.0001

HPV16 L1 VLPs

100 80 60 40 20 0

PfTrx-16L2

PfTrx-L2 mix


HPV16 L1 VLPs

Figure 2 (E)

HPV33 L1 PBNA p < 0.0001

p < 0.0001

100

% Neutralization

% Neutralization

p = 0.045

HPV33 L2 PBNA

80 60 40 20 0

PfTrx-16L2

PfTrx-L2 mix

HPV16 L1 VLPs

100 80 60 40 20 0

PfTrx-16L2

PfTrx-L2 mix


HPV16 L1 VLPs

Challenge with HPV16 PSV

(A) average radiance (p/s/cm2/sr)

Figure 3

p = 0.001

100,000

p = 0.001

p = 0.001

50,000 50,000 40,000 30,000 20,000 10,000 0

neg ctrl

PfTrx-16L2 358 1,735 698 1,900 1,736

PfTrx-L2 mix HPV16 L1 VLPs 192 1,872 2,925 1,154 1,035

5,200,000 4,300,000 1,700,000 2,700,000 2,300,000


average radiance (p/s/cm2/sr)

(B)

p = 0.028

600,000

p = 0.008

p = 0.0193

300,000 300,000 250,000 200,000 150,000 100,000 50,000 0

neg ctrl

PfTrx-16L2

PfTrx-L2 mix HPV16 L1 VLPs

72 164 185 321 - © 2015 American Downloaded from cancerpreventionresearch.aacrjournals.org on- August 12, 2015. Association for

Cancer Research.

Figure 3


(C) average radiance (p/s/cm2/sr)

ns

p = 0.0193

p = 0.0007

600,000 350,000 100,000 100,000 75,000 50,000 25,000 0

neg ctrl

PfTrx-16L2 67 110 77 65

PfTrx-L2 mix HPV16 L1 VLPs 82 92 60

-


(D) average radiance (p/s/cm2/sr)

ns

ns

p = 0.028

16,000 10,000 4,000 4,000 3,000 2,000 1,000 0

neg ctrl

PfTrx-16L2

PfTrx-L2 mix HPV16 L1 VLPs

287 189 442 400 808 1,592 375 449 Downloaded from cancerpreventionresearch.aacrjournals.org351 on August 12,378 2015. © 2015- American Association for

Cancer Research.

Figure 3


(E) average radiance (p/s/cm2/sr)

p = 0.0047

ns

p = 0.008

250,000 200,000 150,000 100,000 50,000 50,000 25,000 0

neg ctrl

PfTrx-16L2 68 104 -

PfTrx-L2 mix HPV16 L1 VLPs 124 495 105 426

-

(F) Challenge with HPV51 PSV Image Min = -34574 Max = 1.9576e+06 p/sec/cm^2/sr

non-immunized (neg ctrl)

Image Min = -21013 Max = 3.234e+06 p/sec/cm^2/sr

PfTrx-16L2 immunized

6


PfTrx-L2 mix immunized


HPV16 L1 VLP immunized

6

6

4

4

M Ma p

6 6

2

x10

6

x10

4

x10

6

x10

4

2

2

6 0

0

Click # AB20121211100646 Di, 11. Dez 2012 10:06:59

bkg sub flat-fielded cosmic

Expt Number: Animal Number: 1,2,3,4,5

Click # AB20121211102456 Di, 11. Dez 2012 10:25:09 Bin:M (8), FOV25, f1, 1m Filter: Open Camera: IVIS 13203, SI620EEV

Color Bar Min = 10000 Max = 7.6e+06


bkg sub flat-fielded cosmic

Color Bar Bin:M (8), FOV25, f1, 2m Min = 10000Filter: Open Camera: IVIS 13203, SI620EEV Max = 7.6e+06

0

0


2

Expt Number: Animal Number: 1,2,3,4,5

bkg sub flat-fielded cosmic Click # AB20121211103609 Di, 11. Dez 2012 10:36:22 Bin:M (8), FOV25, f1, 1m Filter: Open Camera: IVIS 13203, SI620EEV

C M Ma

bkg sub flat-fielded cosmic Expt Number: Animal Number: 1,2,3,4,5


Click # AB20121211105431 Di, 11. Dez 2012 10:54:44 Bin:M (8), FOV25, f1, 1m Filter: Open Camera: IVIS 13203, SI620EEV

bkg flatcosm Expt Number: Animal Number: 1,2,3,4,5

Infection with HPV16 PSV

(A) average radiance (p/s/cm2/sr)

Figure 4

60,000

p = 0.0171

p = 0.0435

p = 0.0466

1,402

1,846

2,700,000

PfTrx-16L2

PfTrx-L2 mix

HPV16 L1 VLPs

50,000 40,000 30,000 20,000 10,000 0

PfTrx

passively transferred sera


(B) average radiance (p/s/cm2/sr)

ns

p = 0.0194

p = 0.0295

100,000 50,000 50,000 40,000 30,000 300

20,000 10,000 0

PfTrx

PfTrx-16L2

PfTrx-L2 mix

HPV16 L1 VLPs

passively transferred sera Association for Downloaded from cancerpreventionresearch.aacrjournals.org on August 12, 2015. © 2015 American Cancer Research.

Figure 4


(C) average radiance (p/s/cm2/sr)

p = 0.0295

p = 0.0256

ns

250,000 200,000 150,000 100,000

81 126

50,000 0

PfTrx

PfTrx-16L2

PfTrx-L2 mix

HPV16 L1 VLPs



(D) average radiance (p/s/cm2/sr)

p = 0.0016

p = 0.0016

ns

10,000 8,000 6,000 4,000 2,000

357

442

0

PfTrx

PfTrx-16L2

PfTrx-L2 mix

HPV16 L1 VLPs

passively seraAmerican Association for Downloaded from cancerpreventionresearch.aacrjournals.org on Augusttransferred 12, 2015. © 2015 Cancer Research.

Figure 4


(E) average radiance (p/s/cm2/sr)

p = 0.0451

p = 0.0016

ns

300,000 160,000 20,000 20,000 15,000

125

10,000 5,000

211

0

PfTrx

PfTrx-16L2

PfTrx-L2 mix

HPV16 L1 VLPs




Robust in vitro and in vivo neutralization against multiple high-risk HPV types induced by a thermostable thioredoxin-L2 vaccine Hanna Seitz, Lis Ribeiro-Müller, Elena Canali, et al. Cancer Prev Res Published OnlineFirst July 13, 2015.

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