Optimization of RG1-VLP vaccine performance in mice with novel TLR4 agonists

Abstract

Current human papilloma virus (HPV) vaccines provide substantial protection against the most common HPV types responsible for oral and anogenital cancers, but many circulating cancer-causing types remain that lack vaccine coverage. The novel RG1-VLP (virus-like particle) vaccine candidate utilizes the HPV16-L1 subunit as a backbone to display an inserted HPV16-L2 17–36 a.a. “RG1” epitope; the L2 RG1 epitope is conserved across many HPV types and the generation of cross-neutralizing antibodies (Abs) against which has been demonstrated. In an effort to heighten the immunogenicity of the RG1-VLP vaccine, we compared in BALB/c mice adjuvant formulations consisting of novel bacterial enzymatic combinatorial chemistry (BECC)-derived toll-like receptor 4 (TLR4) agonists and the aluminum hydroxide adjuvant Alhydrogel. In the presence of BECC molecules, consistent improvements in the magnitude of Ab responses to both HPV16-L1 and the L2 RG1 epitope were observed compared to Alhydrogel alone. Furthermore, neutralizing titers to HPV16 as well as cross-neutralization of pseudovirion (PsV) types HPV18 and HPV39 were augmented in the presence of BECC agonists as well. Levels of L1 and L2-specific Abs were achieved after two vaccinations with BECC/Alhydrogel adjuvant that were equivalent to or greater than levels achieved with 3 vaccinations with Alhydrogel alone, indicating that the presence of BECC molecules resulted in accelerated immune responses that could allow for a decreased dose schedule for VLP-based HPV vaccines. In addition, dose-sparing studies indicated that adjuvantation with BECC/Alhydrogel allowed for a 75% reduction in antigen dose while still retaining equivalent magnitudes of responses to the full VLP dose with Alhydrogel. These data suggest that adjuvant optimization of HPV VLP-based vaccines can lead to rapid immunity requiring fewer boosts, dose-sparing of VLPs expensive to produce, and the establishment of a longer-lasting humoral immunity.

INTRODUCTION

Prevention of HPV-etiologic oral and anogenital cancers through broad and consistent HPV vaccination programs has achieved a great degree of success. Current vaccines are based on noninfectious VLPs of the major L1 capsid protein that contain no nucleic acid material. FDA initially licensed bivalent and quadrivalent versions of these vaccines which succeeded in reducing infection and subsequent cervical intraepithelial neoplasia (CIN) for the HPV16/18 types that are responsible for 70–80% of HPV-related cancers. However, the HPV type coverage by the most recently FDA-approved nonavalent vaccine, Gardasil-9, only extends protection to 90% of HPV-positive cancers, and thus the need for continued cervical screening cannot be eliminated (1). Further addition of new L1-VLP species to the nonavalent vaccine would continue to increase manufacturing cost-of-goods (COG) with only small incremental improvements in protection coverage over the general population. Therefore, an HPV vaccine that eliminates the need for further increasing the number of L1-VLP populations while extending coverage to more oncogenic genotypes through advantaging a new type of vaccine platform would be a substantial improvement on existing HPV vaccines.

HPV infection occurs through viral engagement of heparin sulfate proteoglycans and laminin on the surface of basal keratinocytes, leading to conformational changes in the capsid and internalization of virions into the endosomal pathway (2). These structural changes expose the minor capsid protein L2 and permit its cleavage by the host-derived convertase furin, a step required for endosomal escape of the viral genome to the cell nucleus. Importantly, the furin cleavage also renders the RG1 protective epitope accessible to neutralizing antibody. This region of L2 demonstrates significant sequence conservation among high risk HPVs, and antisera specific to linear epitopes in the N-terminal HPV16-L2 region have demonstrated cross-neutralization activity directed against multiple HPV subtypes (3–4), a phenomenon not achievable with the L1 capsid subunit (5–7). In particular, the L2 amino acid-region 17–36 has a high degree of conservancy between types and is named RG1 due to recognition by the RG1 mAb (8). However, L1 VLP and native virions are highly immunogenic as compared to linear L2 epitopes, raising concerns about the potency and longevity of L2-specific immunity.

Diverse vaccine configurations have been developed that incorporate protective and cross-neutralizing L2 epitopes (reviewed in (9)). To take advantage of the naturally immunogenic nature of a closed-packed surface L2 array while retaining the well-established potency of the L1 subunit, a VLP vaccine was engineered to express the RG1 HPV16-L2 epitope inserted within the DE loop of each of 360 HPV16-L1 subunits arranged into pentameric capsomers, which spontaneously assemble into VLPs (10). This RG1-VLP vaccine adjuvanted with aluminum salts has demonstrated robust immunity in mice and the induction of cross-neutralization to many mucosal high-risk HPV types, including HPV18 and HPV39, mediated by L2-specific Abs (11). However, Gardasil, adjuvanted only with aluminum hydroxyphosphate sulfate, has demonstrated reduced % seropositivity, lower IgG titers (12), and decreased generation of neutralizing Abs to non-vaccine HPV types (HPV31/45) in human subjects compared to Cervarix (13), which employs a combination adjuvant consisting of Alhydrogel and the TLR4 agonist monophosphoryl lipid A (MPLA). This suggests that the TLR4 adjuvant might also contribute to breadth of cross-protection by enhancing the strength of the response, including to subdominant cross-reactive epitopes.

Bacterial enzymatic combinatorial chemistry (BECC) is a biosynthetic method that can be used to produce lipid A mimetics quickly and efficiently (14). The resulting TLR4 agonists have immunostimulatory properties that are advantageous for use as adjuvants and can be manipulated to initiate a targeted immune response. BECC involves heterologously expressing a wide variety of lipid A-modifying enzymes (acyltransferases, deacylases, phosphatase, and/or glycosyl-transferases) obtained from many different Gram-negative bacterial backgrounds in a host bacterial background. BECC molecules have already demonstrated robust adjuvant activity in mice as compared to Alhydrogel for the Yersinia pestis antigen rF1-V to increase anti-rF1-V IgG and IgG2c titers and to protect from lethal Y. pestis challenge (15).

In BALB/c mice, the novel RG1-VLP vaccine when combined with Alhydrogel has demonstrated strong induction of HPV16-L1- and HPV16-L2-specific Abs as well as cross-neutralization activity against other HPV types (11). In an attempt to further optimize the RG1-VLP vaccine and strengthen its ability for cross-type-specific neutralization, this study investigates the combination of BECC438 (15) or BECC470 with Alhydrogel for the ability of the vaccine to induce higher magnitude humoral responses as well as accelerated and longer-lasting immunity.

MATERIALS AND METHODS

BECC molecule synthesis

The previously described BECC438 and BECC470 Yp KIM6+ strains (14–15) (Fig. 1) were grown in shaking culture at 26 °C for 18 h, within which time the culture reached an OD600 of 1.0–1.4. After pelleting bacteria from the liquid culture, lipooligosaccharide was extracted from the pellet as previously described using a double hot phenol method followed by three 2:1 (vol/vol) chloroform-methanol washes (14). Mass spectrometry was used to confirm the extracted lipid A structures (Bruker Microflex MALDI-TOF, norharmane matrix, negative ion mode).

Fig. 1:

Chemical structures of BECC470 and BECC438. Both molecules are hexa-acylated with one modification in the acyl-chain arrangement. BECC470 has a secondary C12 addition at the 3’ position while BECC438 has a secondary C16:1 addition at the 2’ position. The 4’ phosphate group is removed in BECC470 making it mono-phosphorylated while BECC438 remains bis-phosphorylated.

Measurement of BECC Binding to Alhydrogel

A reversed-phase high performance liquid chromatography method was developed to analyze the amount of BECC470 in a solution. A standard curve was created from 0.01 mg/ml to 0.9 mg/ml BECC470 (Sup. Fig. 1). Binding isotherms for BECC470 were conducted in PBS at 25 °C. Samples were prepared by mixing BECC470 (nine different concentrations) and Alhydrogel (at 0.2 or 1.0 g/L). Samples were vortexed for 5 sec and equilibrated for at least 4 h at r.t. after mixing. After equilibration, samples were centrifuged (4000 ×g for 10 min), the supernatant collected, and the concentration of BECC470 determined from the standard curve. Three independent isotherms were conducted for each Alhydrogel concentration.

Physicochemical analysis of BECC/alum formulations

The size distribution of Alhydrogel (InvivoGen) was determined using a Malvern Mastersizer 3000 (Malvern Instruments). First, 6 mL of dH₂O was added to the sample chamber and a background measurement was taken. 300 μL of Alhydrogel sample was injected for each measurement (Alhydrogel at 1 g/L) for a total light obscuration of ~4%. A refractive index of 1.57 was used for Alhydrogel in the Mie scattering calculations for the determination of size. Size distributions were reported by number. Each sample is an average of ten instrumental replicates. Three independent sample measurements were performed. Zeta potential measurements were performed using a Malvern Helix (Malvern Instruments). One mL of sample was placed in a plastic disposable capillary cell and zeta potential measured with a 632 nm laser in a 173° backscatter configuration. Each sample is an average of six instrumental replicates. The measurement was performed for three independent samples. Dynamic light scattering (DLS) was performed using a Malvern Helix. A 632 nm laser in a 173° backscatter configuration was used to measure autocorrelation functions. The method of cumulants was used to determine the average size. Custom aluminum cuvettes with quartz windows containing 50 μl of sample held at 25 °C were employed during the sample measurement. Three independent samples were analyzed with five instrumental replicates (10 s collection time) for each sample.

Vaccine preparation

RG1-VLPs (lot PB16076) were manufactured by Paragon Bioservices (under contract with MRIGlobal, Repository Contractor for the National Cancer Institute, Division of Cancer Prevention) and were combined with Alhydrogel at 40 μg/ml RG1-VLPs and 1 mg/ml Alhydrogel and incubated on a rocking platform for 1 h at 4 °C. BECC438, BECC470, and PHAD (Avanti Polar Lipids) were solubilized in 1X Dulbecco’s PBS then sonicated for 15 min and added to VLP/Alhydrogel formulations before 1 h at 4 °C rocking. Gardasil-9^™ (Merck, recombinant 9-valent human papillomavirus vaccine) was used as a positive control comparator at a dose of 2 μg HPV16-L1 VLPs per mouse to normalize to the RG1-VLP dose. After 1 h at 4 °C rocking, immunization preps were equilibrated to room temperature and administered to mice.

In vivo mouse vaccination studies

NCI-Frederick is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the “Guide for Care and Use of Laboratory Animals” (National Research Council; 2011; National Academy Press; Washington, D.C.). 8–10-week-old female BALB/c mice (Jackson) were randomized into groups of 8–10 animals and immunized on days 0, 14, and 28 (2-week intervals) or 0, 21, 42 (3-week intervals). Mice were anesthetized with isoflurane before intramuscular (i.m.) injection into the quadriceps muscle with 50 μl dose volumes. In some cases, submandibular bleeds were performed on days 13/14 and 27/28 and terminal bleeds were conducted via cardiac puncture on isoflurane-anesthetized mice at day 42 or day 56. In some cases, spleens were also harvested on day 42. In order to measure longer-term Ab responses, some mice were not terminated on day 42 but were monitored with submandibular bleeds at days 42, 70, 98, and 125, before receiving a 4^th immunization on day 126, followed by terminal bleeds and spleen harvest 1 week later. No systemic adverse events or injection site reactions were observed.

Mouse blood sera and splenocyte preparation

Terminal bleeds were collected in serum separator tubes (Fisher Scientific) at r.t. and centrifuged at 6000 g for 1.5 min. Cell-free sera were collected and stored at −80 °C. Spleens and popliteal lymph nodes were dissociated using the GentleMACS Dissociator (Miltenyi). Cell suspensions were subsequently washed with HBSS (Gibco) + 5% fetal bovine serum (FBS) (Gibco), lysed to remove red blood cells with ACK Lysing Buffer (Thermo-Fisher), and filtered through 70 μm strainers (BD Biosciences). Cells were counted with the Vi-Cell analyzer (Beckman Coulter) and resuspended in RPMI-1640 (Thermo-Fisher) + 10% FBS.

HPV16-L1 VLP and HPV16-L2 RG1 peptide ELISAs

Mouse sera were subjected to quantitation by ELISA of Abs specific to HPV16-L1 VLPs and to the HPV16-L2 RG1 epitope. For HPV16-L1 Ab quantitation, Maxisorp 96-well plates (Thomas Scientific) were coated with lab-produced HPV16-L1 VLPs at 2.7 μg/ml in coating buffer (1X PBS + 0.2% Proclin 300 (Sigma)) and used within 3–5 days after incubation at 4 °C. For HPV16-L2 RG1 epitope-specific Ab quantitation, NUNC streptavidin-coated 96-well plates (Thermo-Fisher) were coated with 250 ng/ml N-terminal-biotinylated L2 peptide (L2 a.a. 17–36 QLYKTCKQAGTCPPDIIPKV) (JPT) in coating buffer (0.1 M Tris buffer, 0.15 M NaCl, 0.1% Tween 20) at 100 μl/well. ELISA plates were incubated with blocking buffer (4% skim milk, 0.2% Tween 20 in 1X PBS) for 1.5 h then washed 4 times with wash buffer (0.25% Tween 20 in saline buffer) using a BioTek EL405 plate washer. Sera samples were diluted in blocking buffer at 1:2500 (L1) or 1:5000 (L2) dilution and then serially diluted 1:2 on the plate for 7 more wells for a final volume of 100 μl/well. Sera used for standards and positive controls were generated from mice vaccinated with RG1-VLPs + Alhydrogel, and BALB/c naïve mouse sera (Innovative Research) was used for negative controls. Sera samples were incubated for 1 h at r.t., gently shaking (300 rpm), followed by plate washing. The secondary Ab conjugate goat anti-mouse IgG-horseradish peroxidase (HRP) (Sigma) was added to the plates at a dilution of 1:20,000 at a volume of 100 μl/well and plates were incubated again for 1 h at r.t., gently shaking. After washing, freshly prepared TMB solution (KPL), according to manufacturer’s instructions, was added at 100 μl/well and plates were incubated 25 min at r.t. protected from light. Reactions were stopped by the addition of 100 μl/well of 0.36N H₂SO₄. Plate optical density (OD) values were measured at 450/620 nm with a SpectraMax M5 (Molecular Devices) instrument and data processed by SoftMax Pro 6.3 (Molecular Devices). Ab levels, expressed as ELISA units (EU/ml), were then calculated by interpolation of OD values from the standard curve by averaging the calculated concentrations from all dilutions which fell within the working range of the standard curve.

Furin-cleaved PsV-based neutralization assay (fc-PBNA)

As described in Wang et al. (16), LoVo-T cells (ATCC CCL-229, human colorectal adenocarcinoma line expressing SV40 Large T antigen) grown to 70–90% confluency were removed by Trypsin/EDTA treatment and seeded at 7,500 cells/well in a 96-well flat-bottom plate and incubated for 24 h at 37 °C, 5% CO₂. Pre-diluted (1:25) mouse sera samples were serially diluted 4-fold in DMEM + 10% FBS media in another 96-well plate, including positive and negative control samples derived from RG1-VLP/Alhydrogel-vaccinated mice and naïve mice, respectively. Furin-cleaved PsV (fc-PsV) particles (from HPV types 16, 18, 39, 6) were generated according to Wang et al. (17) and were also diluted to pre-determined concentrations (1:1500 for HPV16/39, 1:500 for HPV6, 1:125 for HPV18 based on titration assays), then added to 96-well round-bottom plates followed by equal volumes of serially diluted serum samples. Finally, the plates were incubated for 2 h at 37°C. After incubation, the serum/fcPsV particle mixtures were added to the 96-well flat-bottom plates previously seeded with LoVo T cells, and the plates were then incubated at 37 °C for 72 h, after which cell supernatants were transferred to 96-well Optiplates (Perkin-Elmer) and incubated at 70 °C for 45 min. Optiplates were then incubated on ice for 5 min and centrifuged briefly, before SEAP (secreted alkaline phosphatase) substrate (Caymen Chemical) was added, followed by 30 min incubation at r.t., protected from light. Plates were read on a SpectraMax M5 microplate reader. The PBNA titers are reported as the reciprocal of the dilution that caused a 50% reduction in SEAP activity in comparison to the fcPsV-infected cells without added sera.

ELISPOT

Freshly isolated splenocytes were resuspended in culture medium (RPMI-1640 + 10% FBS) at 5e6/ml. The R&D Systems mouse IFN-ɣ ELISPOT kit was performed according to manufacturer’s specifications. After blocking plates with culture media, stimulations were added to plates including 5 μg/ml HPV16-L1 VLPs, 0.5 μg/ml anti-CD3/anti-CD28 (BD Biosciences), and 1:2000 Cell Stimulation Cocktail (eBioscience) in volumes of 100 μl. Lastly, 2.5e5 splenocytes were added per well in triplicate in a volume of 50 μl and plates were incubated in a 37 °C, 5% CO₂ incubator for 42–48 h. ELISPOT plates were then developed according to manufacturer’s protocol. Plates were washed with the BioTek EL405 plate washer. After a developing step, plates were air-dried for 24 h and then imaged and spots counted on an ImmunoSpot Analyzer (C.T.L.) using ImmunoCapture software.

Flow cytometry analysis

Popliteal lymph nodes were harvested from mice two weeks post 3^rd immunization and processed by GentleMACS instrument. Cells were stained with LIVE-DEAD Fixable Viability Stain 510 (BD Biosciences) for cell viability and then blocked with Fc Block (BD Biosciences). Surface staining was conducted with a cocktail including CD4-PerCP-Cy5.5, B220-APC-Cy7, CXCR5-biotin, (all BD Biosciences) and PD-1-BV421 (BioLegend). Cells were washed and resuspended with APC-streptavidin (BD Biosciences), then permeabilized using the Mouse Foxp3 Buffer Set (BD Biosciences), followed by intracellular staining with Bcl-6-PE (BD Biosciences). Fluorescent signals were detected by flow cytometry using FACSCelesta instrument and FACSDiva (BD Biosciences), and gated populations were further analyzed with FCS Express software v6 (De Novo). CD4+ B220-CXCR5+ PD-1+ cells were quantified as well as Bcl-6 expression of that population.

In vitro characterization of BECC molecules

In vitro characterization of BECC470 was performed as previously described for BECC438 (15). Briefly, ex vivo primary cells were cultured in the presence of BECC molecules or other known TLR4 agonists such as E. coli LPS. Cell culture supernatants were measured for levels of cytokine secretion using a Milliplex MAP assay (Millipore). Human monocyte-derived dendritic cells were also cultured in the presence of BECC470 or TLR4 agonists and the resulting upregulation of surface co-stimulatory markers was measured by flow cytometry (AllCells).

Statistical analysis

Statistical analyses were conducted with GraphPad Prism 7 software using one-way ANOVA nonparametric analysis with the Kruskal-Wallis multiple comparisons test. p < 0.05 was considered significant.

RESULTS

BECC molecules tightly associate with aluminum hydroxide without disrupting particle size

The molecules BEC438 and BECC470 (Fig. 1) were chosen for this study on the basis of robust in vitro activity profiles on ex vivo primary cells from both mice and humans (Sup. Fig. 2) and demonstration of in vivo adjuvant activity in mice in the context of an antigen subunit-based vaccine (15). To quantify the interaction between BECC470 and Alhydrogel, binding isotherms were constructed. Samples were prepared by mixing BECC470 (nine concentrations) with Alhydrogel (at 0.2 or 1.0 g/L). After incubating BECC470 with Alhydrogel, resulting formulations were centrifuged and supernatants analyzed for unbound BECC470 using the standard curve (Sup. Fig. 1). BECC470 bound on Alhydrogel (1.0 g/L and 0.2 g/L) was plotted against total BECC470 added in respective formulations. As shown in Fig. 2A, at 0.2 g/L Alhydrogel concentration, the amount of BECC470 bound on the surface of Alhydrogel followed a linear slope until it reached a plateau at ~0.16 g/L. This result suggests that Alhydrogel becomes saturated with BECC470 at a 5:4 mass ratio (Alhydrogel to BECC470). In contrast, the bound BECC470 did not reach a plateau at the 1.0 g/L Alhydrogel concentration. If the saturation mass ratio of 5:4 is similar for 1 g/L Alhydrogel, we would expect a plateau at 0.8 g/L of bound BECC470 which seems reasonable given these data. The slope of the first five points of each isotherm was used as a measure of binding affinity (BECC470 bound per BECC470 added). For both binding isotherms 0.2 g/L and 1 g/L Alhydrogel, 0.62 g and 0.95 g BECC470 were bound to Alhydrogel per gram of BECC470 added, respectively. Therefore, we chose a concentration of 1.0 g/L Alhydrogel and 0.1 g/L BECC470 because ~95% of the BECC470 is bound to Alhydrogel at this concentration. Furthermore, the ratio of 10:1 mass of Alhydrogel to mass of BECC470 is similar to that used in the FDA-approved adjuvant AS04 and therefore pharmaceutically relevant.

Fig. 2:

Physiochemical characterization of BECC470 interaction with Alhydrogel. (A) Binding isotherms of BECC470 bound to 1 g/L and 0.2 g/L Alhydrogel were constructed using a Malvern Mastersizer. The error bars represent the standard deviation of three measurements. (B) Size distribution of particles formed by association of Alhydrogel at 1 g/L, alone in dH₂O or 1X PBS, and Alhydrogel added to BECC470 (0.1 g/L). The error bars represent the standard deviation of three replicates.

The size distribution of Alhydrogel in dH₂0 spanned from 0.5 to 10 μm (Fig. 2B). The average size of Alhydrogel in dH₂0 was found to be 1.05 μm, with a marginal increase to 1.66 μm for Alhydrogel in PBS and an intermediate size of 1.44 μm for Alhydrogel with BECC470 (Table 1). Alhydrogel is known to exchange with phosphate groups from other molecules which results in a general increase in negative charge. The zeta potential of Alhydrogel shifted from 12.3 mV in water to −13.7 mV in PBS. BECC470 formed particles with a diameter of ~120 nm, similar to the 90 nm size reported for the aqueous formulation of a MPLA synthetic derivative glucopyranosyl lipid-A (GLA) (18). The BECC470 particles had a zeta potential of −7.9 mV, with the negative charge coming from the phosphate head-groups. When BECC470 was added to Alhydrogel in PBS, the zeta potential decreased further to −16.6 mV. These analyses demonstrate that BECC470 strongly associates with Alhydrogel particles in solution, has a negligible effect on alum particle size, and retains the negative charge of both molecules.

Table 1:

Average particle size and zeta potential of BECC470 complexed with Alhydrogel. Solutions of Alhydrogel in dH₂O or PBS, BECC470 in PBS, or a mixture of BECC470 and Alhydrogel in PBS at the concentrations indicated were analyzed on a Malvern Mastersizer 3000 and a Malvern Helix. Data reported are means of three independent performance runs. Error is the standard deviation of three replicates.

Sample	Zeta Potential (mV)	Number Average Size (μm)
BECC470 (1 g/L) in PBS	−7.9 ± 1.0	0.121 ±0.006*
Alhydrogel (1 g/L) in dH20	12.3 ± 0.2	1.05 ± 0.01
Alhydrogel (1 g/L) in PBS	−13.7 ± 0.9	1.66 ± 0.01
Alhydrogel (1 g/L) + BECC 470 (0.1 g/L) in PBS	−16.6 ± 1.0	1.44 ± 0.03

^*The size of BECC470 in PBS was determined by DLS while the size for Alhydrogel-containing samples were determined by static light scattering.

Enhanced anti-L1/L2 Ab levels and cross-neutralization titers with BECC molecules as adjuvants

To investigate the adjuvant properties of BECC/Alhydrogel (alum) combination, we utilized RG1-VLPs (11). In the current study, BALB/c mice were immunized intramuscularly three times with 2-week intervals between injections with RG1-VLPs and 50 μg Alhydrogel (alum) either alone or in addition to the synthetic TLR4 agonist PHAD or the BECC molecules (BECC438, BECC470). Sera were analyzed for Abs specific to HPV16-L1 and L2 via ELISA and were additionally tested for neutralizing titers against HPV16/18/39/6 by fc-PBNA. Supplementing alum with PHAD yielded greater L1 ELISA Ab levels compared to alum alone, but the additions of BECC438 or BECC470 achieved higher L1 geomeans and statistical differences over VLPs alone compared to PHAD; BECC470 was the only agonist to achieve statistical relevance compared to VLPs + alum (Fig. 3A). Addition of BECC438 or BECC470 also elevated levels of Abs specific to the L2 RG1 epitope compared to VLPs + alum, with a 2-fold increase in geomeans (Fig. 3B). BECC molecules utilized without an alum vehicle were substantially less active and did not significantly boost responses compared to VLPs alone (Sup. Fig. 3). In some cases, we observed an inverse TLR4L (TLR4 ligand) dose correlation with the higher dose exerting lower adjuvant activity, which has been observed with some TLR agonists administered in vivo, indicative of a negative regulatory feedback loop that may be mediated by TLR signaling pathway adaptor molecules (19).

Fig. 3:

Enhancement of RG1-VLP-specific humoral immunity in the presence of BECC molecules + Alhydrogel. Mice were immunized i.m. with 2 μg RG1-VLP alone or adjuvanted with 50 μg alum +/− 25, 50 μg PHAD, 25, 50 μg BECC438, 25, 50 μg BECC470, or Gardasil-9 on days 0, 14, 28 and peripheral blood sera samples derived on day 42. (A–B) Sera samples were tested for HPV16-L1- and HPV16-L2 RG1-specific IgG via ELISA. (C–F) Sera samples were analyzed for neutralizing titers via fc-PBNA specific for PsV16-, PsV18-, PsV39-, PsV6-SEAP. (F) Numbers of responding mice with detectable titers of PsV6-neutralizing activity are indicated. Data are reported as geometric means +95% CI. Statistical comparisons are between VLPs alone and all groups or between alum and all groups (upper tier for (A)) and were generated using one-way ANOVA nonparametric analysis with the Kruskal-Wallis multiple comparisons test. ns, not significant (p > 0.05); *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

An in vitro furin-dependent cell-based neutralization assay was developed to more closely resemble in vivo infectivity (17). Titers of neutralization Abs that prevent the in vitro infection of LoVo-T cells by SEAP-expressing HPV16 PsVs were substantially higher when the RG1-VLP vaccine had been adjuvanted with alum + BECC molecules and in particular with BECC470 (Fig. 3C). Cross-neutralization Abs elicited by the RG1-VLP vaccine were demonstrated by the presence of titers specific to HPV18 and HPV39. Adjuvantation with Alhydrogel raised the levels of HPV18- and HP39-neutralizing titers compared to VLPs alone, but the increase did not reach statistical significance (Fig. 3D–E). However, the presence of BECC molecules further increased the titers of both Ab species and achieved statistical relevance. Finally, cross-neutralizing titers to HPV6 proved the most difficult HPV substrain for TLR4L-augmented Alhydrogel to generate substantial boosting of responses (Fig. 3F). The higher number of nonresponding mice when detecting neutralizing Abs to HPV6 may be due to starker sequence differences between the HPV16-L2 RG1 epitope and the equivalent epitope within HPV6-L2.

Optimization of RG1-VLP vaccine with BECC470 allows for VLP dose-sparing and decreased number of immunizations

The RG1-VLP vaccine requires two additional boosts after the initial prime vaccination to yield substantial levels of L1 and L2-specific Abs when adjuvanted with Alhydrogel alone (10). To determine whether the addition of BECC438 and BECC470 could accelerate the kinetics of the humoral response to RG1-VLP vaccine, we measured Ab levels to both L1 and L2 at earlier timepoints, 2 weeks after first immunization (day 14) and 2 weeks after the second immunization (day 28) as well as 2 weeks after the third immunization (day 42). Both BECC438 and BECC470 adjuvants substantially elevated L1 and L2 ELISA Ab levels compared to alum alone after only 2 immunizations (Fig. 4A–B). Additionally, the presence of BECC470 enabled the RG1-VLP vaccine with 2 vaccinations to achieve L1 and L2 Ab levels equivalent to or superior than L1 and L2 Ab levels achieved by 3 vaccinations with alum alone (dotted line). BECC470 also accelerated the appearance of neutralizing Ab titers to HPV16/18 PsVs, achieving superiority to alum alone by day 28 (Fig. 4C–D). These data emphasize the potential of a combined BECC/alum adjuvant allowing for fewer booster shots of a L1-VLP-based vaccine. Additionally, Fig. 5A–B indicates that reducing the RG1-VLP dose to 1 μg or even 0.5 μg while adjuvanted with BECC470/alum resulted in L1 and L2 ELISA Ab levels that were comparable (L1) or superior (L2) to levels achieved by alum alone with the full 2 μg VLP dose, indicating that BECC470 does allow for VLP dose-sparing. Neutralization assays run on the same sera samples revealed that induction of HPV16, HPV18, HPV39, but not HPV6, neutralizing Ab titers by 0.5 μg RG1-VLPs + BECC470/alum was also comparable to titers achieved by 2 μg RG1-VLPs + alum alone (Fig. 5C–F). Such robust humoral responses arising from only 25% of the standard mouse VLP dose underlines the benefits in dose-sparing and COG-savings that could be achieved with an appropriately potent adjuvant combination.

Fig. 4:

BECC molecules + Alhydrogel accelerate the appearance of L1/L2 Ab levels as well as HPV16/18-neutralization titers. Mice were immunized i.m. with 2 μg RG1-VLP alone or adjuvanted with 50 μg alum +/− 25 μg PHAD, 25 μg BECC438, 25 μg BECC470, or Gardasil-9 on days 0, 14, 28 and peripheral blood sera samples derived on days 14, 28, 42. (A–B) Sera samples were tested for HPV16-L1- and HPV16-L2 RG1-specific IgG via ELISA. Statistical comparisons are between VLPs alone and adjuvanted groups for each time point, absence of designation indications no statistical difference. Dotted line represents Ab levels with alum alone at day 42 for comparison to earlier time points. (C–D) Day 14/28 sera samples were analyzed for neutralizing titers via fc-PBNA specific for PsV16-, PsV18-SEAP. Data are reported as geometric means +95% CI. Statistical comparisons are between alum and BECC/alum groups and were generated using one-way ANOVA nonparametric analysis with the Kruskal-Wallis multiple comparisons test. ns, not significant (p > 0.05); *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Fig. 5:

Immunization with reduced VLP doses achieves optimal L1/L2 Ab levels and HPV-neutralizing titers when adjuvanted with Alhydrogel/BECC470. Mice were immunized i.m. with 0.5/1/2 μg RG1-VLP alone or adjuvanted with 50 μg alum +/− 25 μg BECC470, or Gardasil-9 on days 0, 14, 28 and peripheral blood sera samples derived on days 14, 28, 42. (A–B) Sera samples were tested for HPV16-L1- and HPV16-L2 RG1-specific IgG via ELISA. Statistical comparisons are between VLPs alone and adjuvanted groups for each time point, absence of designation indications no statistical difference. (C–F) Day 42 sera samples were analyzed for neutralizing titers via fc-PBNA specific for PsV16-, PsV18-, PsV39-, PsV6-SEAP. Data are reported as geometric means +95% CI. Statistical comparisons are between VLPs alone and all groups or between alum and all groups (upper tier for (C)) and were generated using one-way ANOVA nonparametric analysis with the Kruskal-Wallis multiple comparisons test. ns, not significant (p > 0.05); *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

BECC470 enables long-lasting enhanced Ab responses as well as robust memory L1-specific T cell responses

Although it is clear that neutralizing Abs that inhibit HPV infection are critical for a protective response, it is less clear what role HPV-specific T cell responses may play. Alum-formulated vaccines are historically poor for induction of T cell responses, potentially due to the activation of the inflammasome pathway which does not optimally prime dendritic cells to activate T cells (20). Combining either BECC molecule with alum in our vaccine formulation led to robust HPV16-L1 VLP-specific T cell responses as measured by ELISPOT (Fig. 6), while alum alone was typically poor. An additional boosting effect on this T cell response could be observed in mice that received a 3^rd boost approximately 3 months after the 2^nd boost (on day 42) (Sup. Fig. 4), indicating the presence of long-term memory T cells. Bringing this additional arm of the immune response to bear could lead to higher vaccine efficacy by developing a cytotoxic T cell response specific for HPV-infected cells.

Fig. 6:

Induction of robust IFN-ɣ responses after BECC molecule supplementation of Alhydrogel. Mice were immunized i.m. with 2 μg RG1-VLP alone or adjuvanted with 50 μg alum +/− 25, 50 μg PHAD, 25, 50 μg BECC438, 25, 50 μg BECC470, or Gardasil-9 on days 0, 21, 42 and peripheral blood sera samples derived on day 56. Spleens were harvested on day 56 and splenocytes restimulated in vitro with HPV16-L1 VLPs for 48 h an SFUs (spot-forming units)/1e6 cells analyzed via ELISPOT. Data are reported as geometric means +95% CI. Statistical comparisons are between alum-adjuvanted group and all groups and were generated using one-way ANOVA nonparametric analysis with the Kruskal-Wallis multiple comparisons test. ns, not significant (p > 0.05); *, p < 0.05; **, p < 0.01; ***, p < 0.001.

The most effective vaccines are able to induce long-lasting immunity that is characterized by strong memory T and/or B cell responses and are protective long after the initial vaccination. To determine whether the addition of BECC470 resulted in such long-lasting memory Ab responses, we monitored L1 ELISA Ab levels for 3 months after the third immunization at day 42. Fig. 7A shows that although total L1 Ab levels slowly wane over time, the boosted magnitude delivered by the presence of BECC470 compared to alum alone also continues over time, and although gradually losing statistical significance, still retains a 2.3-fold difference in L1 titers at day 125 (day 195 of the animal’s lifespan). To further pursue the question of establishment of memory, the presence of T follicular helper (Tfh) cells in the spleen and popliteal lymph nodes (popLNs) was investigated, as Tfh cells are known to be critical drivers of memory B cell and plasma cell differentiation (21–22). At day 42, 2 weeks post third immunization, a substantial enhancement of the percentage of Tfh cells within the CD4 T cell compartment of both spleens and mLNs was observed in the BECC470/alum-adjuvanted mice, while the increase in Tfh cells in the alum alone group was significantly less robust (Fig. 7B–C). Therefore, the presence of BECC470 clearly improved the L1-specific B cell memory response, probably through robust proliferation of the Tfh subset within lymphoid centers.

Fig. 7:

BECC470 induces T follicular helper cells in lymphoid compartments as well as a sustained elevation of the L1 Ab response over several months. Mice were immunized i.m. with 2 μg RG1-VLP adjuvanted with 50 μg alum +/− 25 μg BECC470on days 0, 14, 28 and peripheral blood sera samples derived on days 14, 28, 42, 70, 98, 125. Some mice were sacrificed on day 42 and splenocytes and popliteal LN cells were analyzed for Tfh content via FACS. (A–B) Sera samples were tested for HPV16-L1-specific IgG via ELISA. Data are reported as geometric means +95% CI. Statistical comparisons were generated using nonparametric Mann-Whitney t-test. ns, not significant; p > 0.05; *, p < 0.05; **, p < 001. (B–C) Splenocytes and popliteal LNs were stained for presence of CD4⁺ CXCR5⁺ PD-1⁺ Tfh cells. Statistical comparisons were between VLPs alone and other adjuvanted groups and were generated using one-way ANOVA nonparametric analysis with the Kruskal-Wallis multiple comparisons test. ns, not significant (p > 0.05); *, p < 0.05; ***, p < 0.001.

DISCUSSION

Although effective at providing protection against HPV infection as well as onset of HPV-related cervical and oropharyngeal cancers, the currently licensed HPV VLP-based vaccines Gardasil and Cervarix would benefit from optimization. Although RG1-VLPs show significant promise as an approach to achieve broad immunity against high risk HPV genotypes using only a single antigen, the response to the L2 component may not be optimal in the current Alhydrogel adjuvant. The RG1 epitope of RG1-VLP is highly immunogenic in mice (Fig. 3B, (23)) with substantial levels of L2-specific Abs detectable after only 1 immunization with Alhydrogel (Fig. 4B); however, substantial L1 Ab levels require 2 immunizations to discern, and 3 immunizations for optimal production (Fig. 4A), while optimal neutralizing Ab titers to HPV16 and HPV18 also require 3 immunizations for high magnitude responses (Fig. 4C–D).

The TLR4 agonist MPLA was among the first of several families of TLR ligand-sourced compounds to achieve FDA approval for use in Cervarix and the HBV vaccine Fendrix; however, few cGMP-grade TLR4 agonists are available. Although achieving FDA licensure, MPLA is a heterogenous preparation of multiple species of lipid A structures or congeners that can vary in composition between batches. A very few synthetic alternatives, such as PHAD, are commercially available and have yielded successful results in clinical trials (24–25). The invention of the BECC system has led to the generation of dozens of distinct TLR4Ls through manipulation of the expression of enzymes involved in the generation of lipid A within transfected bacteria (14). Examples of these compounds have already proven vigorous adjuvant activity in mouse models of Yersinia pestis lethal challenge (15), which described a vaccine platform of soluble rF1-V antigen + aluminum salt adjuvant. The current study proves that VLP-based vaccines can also benefit from BECC adjuvant activity when paired with Alhydrogel.

Current ACIP recommendations for the standard of care HPV vaccine in the U.S., Gardasil-9, is a 3-dose series administered with 1-month and 5-month intervals between doses 1–2 and doses 2–3, respectively, if starting at age 15 or older. Two doses has been determined sufficient, however, if the vaccination regime starts earlier, by age 9. Major clinical trials are underway to examine the value of a single dose of Cervarix or Gardasil-9. Decreasing the recommended doses for patients of all ages would substantially cut down on COG of the vaccine and increase affordability. Furthermore, maintaining compliance, necessary for the establishment of long-lasting immunity, could also be achieved at higher rates with a fewer-dose schedule. The WHO has recommended 2-dose schedules for <15 year-olds, but a 3-dose regime remains recommended for vulnerable patient populations such as older age cohorts (26–28). In the current study, a substantial boosting effect provided by the 3^rd immunization of the alum vaccine was observed in both L1 and L2 Ab levels (Fig. 4–5) as well as neutralizing Ab titers to HPV16 and HPV18 (Fig. 3–4), as compared to levels after only 2 immunizations. Optimizing the alum adjuvant formulation with BECC470, however, achieved L1 and L2 Ab levels after 2 doses that was equivalent (L1) or superior (L2) to levels achieved with 3 doses of the alum vaccine (Fig. 4A–B), and neutralization titers to HPV16/18 were also substantially enhanced after only 2 doses (Fig. 5C–D). This demonstrates the benefits of optimizing the adjuvant formulation for any VLP-based HPV vaccine which could lead to reduced dose-schedules, higher compliance, improved responses in immunocompromised subjects, and reduced COG.

Further amelioration of COG could be achieved by lowering the amount of the HPV VLP component in the vaccine. Potent adjuvant formulations have been noted not only for their strong immunogenic potential but also their capacity to retain robust immunogenicity even at lower antigenic doses (29–30). We observed that supplementing Alhydrogel with BECC470 resulted in substantial VLP dose-sparing effects. Levels of Ab responses to both HPV16-L1 and HPV16-L2 achieved at 2 μg RG1-VLP with Alhydrogel alone were matched (L1) or exceeded (L2) at 25% of the standard VLP dose when Alhydrogel was combined with BECC470 (Fig. 5). Neutralization titers against HPV16/18/39 at the 0.5 μg VLP dose were also achieved with the addition of BECC470 to alum that were comparable to titers yielded by 2 μg VLPs + Alhydrogel alone. These data indicate BECC470 would further COG savings by allowing dose-sparing of the VLP component of the vaccine, helping mitigate this significant cost outlay for VLP-based vaccines.

Interestingly, in this murine model of HPV vaccination, BECC470 consistently outperformed BECC438 in adjuvant capabilities. This observed difference highlights the importance of structural changes of TLR4Ls to their ability to trigger signaling downstream of the TLR4/MD2 receptor complex. Although both MPLA and BECC470 are mono-phosphorylated, they differ by position of phosphate removal: 1’-position for MPLA and 4’-position for BECC470, and instead of the 2’ secondary 16-C acyl-chain addition with one unsaturation of BECC438, BECC470 has a 3’ secondary 12-C acyl-chain addition. This combination of phosphate removal and relative asymmetry of the BECC470 molecule may allow for formation and stabilization of a TLR4 receptor signaling complex that is particularly advantageous to promoting the induction of protective immunity without an overabundance of reactogenic responses (31).

While current HPV vaccines have enjoyed remarkable success at reducing rates of HPV infection and subsequent incidence of HPV-related cancers, there remains a need for improved vaccines that will lower COG, reduce the vaccine schedule, and amplify the humoral response. Our data indicate that supplementation of Alhydrogel with a BECC molecule can optimize the activity and potency of the novel RG1-VLP vaccine by enhancing the magnitude of the Ab response, by allowing for VLP dose sparing, by accelerating the appearance of protective levels of neutralizing Abs and thus allowing for a shorter vaccine schedule, and by promotion of longer-lasting Ab responses. Given the superiority displayed by BECC molecules over the standard TLR4L MPLA in terms of adjuvant activity, scale of synthesis, purity, and chemical stability, further investigation of adjuvanting RG1-VLPs with a combination adjuvant preparation of aluminum hydroxide + BECC molecules may lead to the development of a lost-cost, high efficacy next-generation HPV vaccine and represent an AS04 biosimilar adjuvant platform.

HIGHLIGHTS.

• BECC TLR4L compound added to Alhydrogel improves humoral responses to RG1-VLP vaccine.

• VLP dose-sparing and reduction in injection frequency were achieved with addition of BECC.

• Long-lasting effects on HPV-L1 Ab response induced with BECC compound.

ABBREVIATIONS

Ab

antibody

BECC

bacterial enzymatic combinatorial chemistry

CIN

cervical intraepithelial neoplasia

COG

cost of goods

ELISA

enzyme-linked immunosorbent assay

ELISPOT

enzyme-linked immunospot assay

FBS

fetal bovine serum

fc-PBNA

furin-cleaved pseudovirion-based neutralization assay

HPV

human papilloma virus

HRP

horseradish peroxidase

i.m.

intramuscular

MPLA

monophosphoryl lipid A

OD

optical density

PHAD

phosphorylated hexa-acyl disaccharide

popLNs

popliteal lymph nodes

PsV

pseudovirion

r.t.

room temperature

SEAP

secreted alkaline phosphatase

SFUs

spot-forming units

Tfh cells

T follicular helper cells

TLR4

Toll-like receptor 4

TLR4L

TLR4 ligand

VLP

virus-like particle