Self-adjuvanting lipoimmunogens for therapeutic HPV vaccine development: potential clinical impact

Summary

The goal of therapeutic human papillomavirus (HPV) vaccines is the induction of cytotoxic T lymphocyte (CTL) immunity against HPV-associated cancers.

Recombinant proteins and synthetic peptides have high safety profiles but low immunogenicity, which limits their efficacy when used in a vaccine. Self-adjuvanting lipid moieties have been conjugated to synthetic peptides or expressed as lipoproteins to enhance the immunogenicity of vaccine candidates. Mono-, di- and tri- palmitoylated peptides have been demonstrated to activate dendritic cells (DCs) and induce robust cellular immunity against infectious diseases and cancer. Recently, a platform technology using the high-yield production of recombinant lipoproteins with TLR2 agonist activity was established for the development of novel subunit vaccines.

This technology represents a novel strategy for the development of therapeutic HPV

vaccines. In this review, we describe recent progress in the design of therapeutic HPV

vaccines using lipoimmunogens.

Keywords

Human papillomavirus Lipopeptide

Toll-like receptor 2

Recombinant lipoimmunogen

Self-adjuvantinged vaccine

Introduction of HPV-associated cancers

Cervical cancer is a malignancy that is closely associated with human

papillomavirus (HPV) infection. More than 500,000 new cases and 275,000 deaths

from cervical cancer occur annually worldwide, as reported by the World Health

Organization (WHO) HPV Information Centre Report [1]. HPV-associated cervical

carcinogenesis occurs over the course of decades, but the mortality rate of cervical

cancer is very high (52%) [2]. HPV16 and 18 are classified as high-risk types that

cause cervical cancer in more than 70% of cases [3]. HPV persistently infects cervical

mucosal epithelia, which leads to cervical intraepithelial neoplasia (CIN) and invasive

cancer after 10-20 years. Based on the proportion of abnormal cells in the epithelium,

CIN can be categorized into grades 1-3 (CIN 1-3). The oncogenic viral proteins HPV

E6 and E7 are detectable in cervical mucosal epithelial cells in patients with early-

stage CIN (CIN 1). These proteins regulate cell cycle and cell death and contribute to

tumorigenesis [4]. The over-expression of HPV E6 and E7 in mucosal epithelial cells

is necessary for cell transformation and the progression of invasive cancer. In addition

to cervical cancer, HPV infection causes other cancers, including cancers of the

external genitalia (vulva, vagina and penis), the anus, the mouth and the oropharynx

[5]. HPV infection is associated with 40%-51% of vulvar cancers, 40%-64% of

vaginal cancers, 36%-40% of penile cancers, 90%-93% of anal cancers, and 12%-

63% of oropharyngeal cancers [6]. HPV infection leads to squamous cell carcinoma

more frequently than adenocarcinoma. Tobacco and alcohol are risk factors for head

and neck squamous cell carcinoma (HNSCC); however, HPV-associated HNSCC has

been identified in 35.6% of oropharyngeal cancers, 23.5% of oral cavity cancers and

24% of laryngeal cancers [7,8]. Therefore, approaches are urgently needed to control and cure cancers that are caused by HPV infection.

Fortunately, prophylactic HPV vaccines have been developed for use in humans.

Current prophylactic vaccines, such as Gardasil and Cervarix, utilize virus-like particles (VLPs) that are formulated with adjuvants. The major component of VLPs is the HPV capsid protein L1, which induces neutralizing antibodies against the virus.

These vaccines have decreased the incidence of CIN and cervical cancer. However, prophylactic vaccines cannot induce efficient CTL responses against the virus in HPV-infected CIN and cancer patients; therefore, prophylactic vaccines have no therapeutic effect and cannot be used in these patients. To address this important public health problem, it is necessary to develop efficient therapeutic HPV vaccines for HPV-infected populations. The HPV E6 and E7 oncoproteins, but not the HPV L1 protein, are over-expressed in transformed cells. Therefore, the HPV E6 and E7 oncoproteins may be candidates for use in therapeutic vaccines to eliminate virally infected cells and to inhibit tumor progression [9,10]. Several different approaches have been reported to induce E6- or E7-specific CTL responses against HPV- associated cancer; however, in this report, we focus on the development of subunit vaccines for the treatment of HPV-associated cancer.

Presentation of immunogens by dendritic cells to prime CTL responses

Dendritic cells (DCs) are specialized antigen-presenting cells that present

antigens and provide immunological signals to regulate T cell activation [11,12]. DCs

can process endogenous and exogenous antigens and present epitopes on major

histocompatibility complex (MHC) class I and class II molecules for CTLs and helper T (Th) cells, respectively [13]. DCs can present exogenous antigens via the cross- presentation pathway, which is an important mechanism for therapeutic tumor vaccines [14,15]. Therefore, DCs can process tumor-associated antigens (TAAs) and present appropriate epitopes to induce antigen-specific anti-tumor CTL activation [16]. However, native proteins are inefficiently cross-presented by DCs and have low immunogenicity due to their reduced CTL activation. To enhance the ability of DCs to prime CTLs, the cross-presentation mechanisms of DCs must be manipulated. Two strategies can be used to improve the cross-presentation ability of DCs.

The first strategy is to increase the quantity of immunogens in the intracellular compartment that may cross-present CTL epitopes to CD8

T cells. Viral and DNA vectors can carry TAA genes into the cytosol to express intracellular antigens.

Intracellular antigens are processed by the proteasome for the identification of CTL

epitopes, which are presented via the MHC I pathway for CTL activation (Fig. 1). In

contrast, exogenous protein/peptide antigens can be conjugated by to antibodies or

carbohydrates to target receptors on DCs, e.g., CD11c, CD32, DC-SIGN and dectin-1,

for enhancing CTL cross-priming via the cross-presentation pathway [14]. Moreover,

charge-modifyingied or palmitoylated peptides that penetrate into the cellular cytosol

are a potential approach for enhancing antigen presentation [17-20]. The second

approach is to activate dendritic cells through innate receptors and to regulate the

cross-presentation process. Toll-like receptors (TLRs) are pattern recognition

receptors (PRRs) that recognize pathogen-associated molecule patterns (PAMPs) to

induce pro-inflammatory cytokines and interferons for cellular innate defenses [21].

TLR agonists stimulate the maturation of DCs, the secretion of cytokines and the up- regulation of co-stimulatory molecules to induce Th cell differentiation and CTL activation [22]. The TLR9 agonist CpG oligodeoxynucleotide enhances IL-12 secretion and promotes antigen cross-presentation by DCs to elicit stronger anti-tumor CTL responses [23]. TLR2 cooperates with TLR1 and TLR6 to recognize triacyl- and diacyl-lipopeptides, respectively [24]. Synthetic TLR2 agonists, such as macrophage- activating lipopeptide-2 (MALP-2), Pam2CSK4 and Pam3CSK4, can induce Th1 immune responses and DC maturation. In addition to the simple lipidated peptides, there are some new approaches have been reported that include lipoamino acid based adjuvants [25-27]. Furthermore, our studies have demonstrated that TLR2 agonist- conjugated lipopeptides activated DCs and enhanced antigen presentation by regulating the antigen-processing pathway [28]. Adjuvants that are targeted to TLRs can activate antigen-presenting cells and increase antigen presentation to CD8

T cells. Accordingly, TLR agonist-conjugated proteins or peptides can be used in therapeutic vaccines against virus-associated cancers.

Self-adjuvanting lipopeptides induce CTL responses

In recent decades, synthetic long peptides conjugated with palmitic acid have

enhanced antibody production and MHC class II presentation [29]. The use of

synthetic palmitoylated peptides is a convenient strategy to conjugate peptides and

palmitic acid for antibody induction [30]. In contrast to the co-adminstration of

palmitic acid lipid moiety and immunogen, pamitoylated peptides are able to co-

deliver immunogen and lipid moiety into the same antigen-presenting cells. The co-

delivery could be uptake more efficiently into antigen presentation pathway and

activate the same antigen-presenting cells. The di- or tri- pamitoylated peptides could

be internalized into antigen-presenting cells though TLR2. Therefore, a physical

linkage between the lipid moiety and immunogen is a rational design for inducing

CTL responses. Furthermore, palmitoylated peptides with cytotoxic T cell epitopes

have been demonstrated to induce CTL responses via cross-presentation [31]. In

addition, natural killer (NK) cells also play an important role in anti-cancer immune

responses that induced by palmitoylated peptides immunization [32]. Palmitoylated

peptide vaccines have been examined for use in several diseases. Table 1 lists the

lipidated proteins/peptides that elicit CTL responses against the hepatitis B virus

(HSVHBV), the hepatitis C virus (HCV), the human immunodeficiency virus (HIV),

HPV, influenza, malaria and Mycobacteria tuberculosis in animal studies or clinical

trials. As a vaccine against hepatitis B, the Theradigm-HBV vaccine consists of a

lipopeptide that contains a CTL epitope (derived from the HBV core antigen), which

is covalently linked with a Th epitope (derived from tetanus toxoid). Additionally, the

lipopeptide is palmitoylated at the N-terminal [33,34]. In clinical trials, Livingston et

al. demonstrated that the Th responses that were induced by the Th epitope enhanced

memory CTL development. The Th cells have been shown that plays an important

roles to enhance anamnestic responses [35,36]. Activation of Th cells provides

cytokines, like IL-2, for fully activation of CTL responses. The engagement of TLR2

by lipoeptptide could activate DCs also lead to enhance Th licensing between CD40

and CD40L interaction for memory CTL differentiation [37]. Incorporation of

autologous Th epitopes into synthetic lipopeptide may strongly increase anamnestic

CTL responses. The use of multiple lipopeptides is another approach to elicit efficient CTL responses, e.g., LIPO-4, LIPO-5 or LIPO-6 against HIV [38-43]. These vaccines against HIV can elicit prolonged CTL responses over 24-48 weeks in healthy recipients and HIV patients. Pam2- and Pam3-linked peptides have been used in vaccines against HCV, HPV and influenza because of their adjuvant effects (Table 1).

These lipopeptides have been used in self-adjuvanting vaccines to stimulate innate immune responses and to improve CTL responses for the reduction of viral titers and tumor size [28,44]. However, the challenges in developing therapeutic cancer vaccines include the need to elicit strong CTL responses against cancer cells and the need to overcome the immunosuppressive effects in the tumor microenvironment. The advantage of mixtures of individual lipidated peptides or lipidated multiple linear CTL epitopes is capable of inducing CTL responses against different antigens.

However, they may only contain well-identified CTL epitopes that could not be applied to each individual. In contrast, the lipidated protein may provide diverged Th or CTL epitopes to overcome the MHC-restricted manner.

Mono-palmitoylated peptides have been demonstrated to effectively elicit

antigen-specific CTL activity against tumors [18,45]. Xu et al. found that

immunization with a mono-palmitoylated peptide without adjuvant could induce

CTLs to kill peptide-pulsed target cells. Furthermore, the mono-palmitoylated peptide

could elicit CTLs in PBMCs from healthy human donors in vitro, which induced

cytotoxicity in HPV E7-expressing tumor cells (Caski cells) [38]. Mono-

palmitoylated peptides have no effect on promoting DC maturation. However,

compared with a non-lipidated peptide, Song et al. demonstrated that mono-

palmitoylated peptides could increase antigen cross-presentation to CD8

T cells via another internalization mechanism in DCs [18]. Andrieu et al. found that mono- palmitoylated Pol

and Nef

peptide-pulsed DCs have different sensitivities for antigen-processing inhibitors, such as monensin, brefeldin A and epoxomicin, which stimulate CTLs [31]. Therefore, these studies indicate that mono-palmitoylated peptides can be internalized and processed by DCs using several mechanisms for CTL activation. In the past two decades, Pam3Cys-conjugated peptides, which are analogous to the immunologically active bacterial lipoprotein, have been used to enhance immune cell activation and to improve immunogenicity [30,46]. The bacterial lipoprotein analogs Pam3CSK4 and Pam2CSK are well-known Toll-like 2 (TLR2) agonists that induce DC maturation. The C16 alkane chain on the Pam2Cys peptide is critical for the engagement of TLR2 and the activation of DCs [47]. Baz et al. demonstrated that both linear (N-terminal attached) and branched (internal lysine residue attached through two serine residues) Pam2Cys-conjugated peptides could induce CTL killing activity and antitumor responses [48]. However, the branched Pam2Cys-conjugated peptide was found to induce stronger CTL responses than the linear Pam2Cys-conjugated peptide. Moreover, synthetic Pam2Cys/Lys and Pam3Cys/Lys peptides that contain CTL epitopes and act as TLR2 agonist-conjugated antigenic peptides can target and activate DCs to induce effective CTL responses without conventional adjuvants (Table 1). The application of lipid-based proteins/peptides in therapeutic HPV vaccines is reviewed in greater detail below.

Induction of CTL responses against HPV-associated cancer

Many different approaches to inducing induce effective CTL responses against HPV-associated cancer have been developed, such as subunit, genetic and DC-based vaccines. The disadvantages and advantages of these approaches have been well reviewed in previous reports [49,50]. In this review, we discuss protein/peptide-based subunit vaccines for HPV-associated cancer. Subunit vaccines, either protein- or peptide-based, contain the immunogenic component of HPV antigens for the induction of CTL responses. For safety reasons, the oncogenic region (the retinoblastoma protein (Rb) binding region) of the HPV E7 oncoprotein is deleted or mutated and purified using a protein expression system for immunization [51]. In contrast to protein immunogens, peptide vaccines are chemically synthesized and contain CTL epitopes for HPV E7 [52,53]. HLA-B18-restricted CTL epitopes, such as HPV E7

, have induced CTL responses in peripheral blood mononuclear cells from healthy donors [45]. Peptide/protein-based vaccines are low cost, easy to manufacture and safe. Therefore, this approach may be used in the development of therapeutic vaccines against HPV-associated cancer. Subunit vaccines are developed and modified to target DCs for improving immunogenicity. These modifications are designed to enhance DC maturation and antigen presentation. Recently, we synthesized lipopeptides that were modified by palmitoylation, and these peptides stimulated DC maturation and antigen presentation, which increased CTL responses against HPV-associated cancer [18,28,54]. Mono-palmitoylated HPV16 E7

peptide-pulsed bone marrow-derived DCs (BMDCs) can induce more antigen-specific

CD8

T cells than non-lipidated HPV16 E7

or HPV16 E7

peptide-pulsed

BMDCs. In an animal study, TC-1 tumor-bearing mice were immunized with 2 x 10

of non-lipidated HPV16 E7

or HPV16 E7

peptide-pulsed BMDCs or mono- palmitoylated HPV16 E7

peptide-pulsed BMDCs. Immunization with mono- palmitoylated peptide-pulsed BMDCs inhibited tumor growth. In contrast, the di- palmitoylated HPV16 E7

peptide stimulated BMDCs to produce more inflammatory cytokines and up-regulated the expression of co-stimulatory molecules through a TLR2-dependent pathway. Immunization with the di-palmitoylated peptide without adjuvant induced tumor regression in TC-1 tumor-bearing mice.

Nevertheless, immunization with the di-palmitoylated peptide induced anti-tumor CTL responses via a TLR2-dependent pathway.

Synthetic lipopeptides for HPV therapeutic vaccines

Lipoimmunogens, such as palmitoylated peptides, have been extensively studied

in pre-clinical and Phase I studies (Table 1). Palmitoylated peptides have been

demonstrated to enhance Th cell responses for humoral immune responses [55]. In

addition, palmitoylated peptides with both CTL and Th epitopes can enhance CTL

responses [56]. As a therapeutic vaccine against HPV-associated cancer, a

palmitoylated CTL epitope of the HPV 16 E7 oncoprotein was used to induce CTLs

in cervical and vaginal cancer patients [57]. Steller et al. found that 4 rounds of

lipopeptide immunization could elicit HLA-A2-restricted CTL responses. The authors

used the lipopeptide to pulse DCs for sensitizing CTLs. These data indicate that

palmitoylated peptides can be effectively presented to CD8

T cells by APCs via the

MHC class I pathway in humans (Fig. 2). When used alone, palmitoylated peptides

can elicit CTL responses; however, appropriate adjuvants are required to induce

tumor regression [58]. To enhance anti-tumor activity, di-palmitoylated peptides (Pam2Cys peptides) with TLR2 ligands agonist activity were developed. A di- palmitoylated peptide with a murine CTL epitope that was derived from the HPV16 E7 oncoprotein was found to have a preventive and therapeutic effect in a TC-1 tumor model [54]. We demonstrated that immunization with the Pam2Cys peptide induced a 2.5- to 3-fold increase in the number of CTLs compared with a non-lipid peptide. In the tumor model study, we found that the Pam2Cys peptide induced preventive and therapeutic effects against TC-1 tumors. In the therapeutic model, the palpable tumor could be inhibited by two rounds of immunization with 10 g of the Pam2Cys peptide at a 7-day interval. Therefore, di-palmitoylated peptide-based vaccines can stimulate BMDC maturation via TLR2/6 and enhance CTL responses to inhibit tumor growth (Table 1). TLR2 engagement activates DC maturation and regulates antigen cross- presentation via the vacuolar pathway through the TLR2 signaling pathway [28].

Figure 2 illustrates the antigen presentation mechanism of di-palmitoylated peptides.

Rab7 up-regulation via TLR2 signaling enhances the immunogenicity of peptide- based vaccines. These results indicate that the incorporation of palmitic acid into antigens is a feasible strategy for vaccine studies. Peptide-based vaccines must utilizeare limited MHC restriction and cannot induce fully functional immune responses against tumors; however, synthetic palmitoylated peptides are useful in preliminary studies of recombinanted lipoimmunogens.

Development of recombinant lipoprotein-based vaccines

Synthetic lipopeptides and biosynthetic recombinant lipoprotein are prepared

from different manufacture methods. Synthetic peptides are prepared by a standard Fmoc-strategy solid-phase peptide synthesis protocol. To synthesize Pam3Cys- conjugated peptides, Fmoc-Pam3Cys-OH is added to the N-terminus of the peptides linked to the resin. Subsequently, the Fmoc group was removed from the synthetic lipopeptide, detached from the resin, and removed all protecting groups (Fig. 3) [59,60]. However, recombinant lipoprotein is lipidated at the lipobox within the signal

sequence by the post-translational modifications. Lipoprotein diacylglyceryl transferase is a critical enzyme to add a diacylgycerol moiety linked by a thioether bond at cysteine in the lipobox to form pro-lipoprotein. Sequentially, lipoprotein signal peptidase cleaves the signal sequence of the lipidated pro-lipoprotein and explores amino-terminal residue of cysteine of the lipobox. Consequently, the N- terminal cysteine residue is attached an amide-liked acyl group by N-acyl transferase in Gram-negative and some Gram-positive bacteria [61]. In order to enhance antigenicity of recombinant immunogens, there are different approaches have been reported to conjugate lipid moiety to immunogensthat include ion-pairing, chelation and conjugation [62-65]. In this review, we are more focus on the recombinant lipoprotein derived from bacterial lipoproteins.

In contrast to peptide vaccination, protein-based vaccination can circumvent the

issue of major histocompatibility complex (MHC)-restricted antigen recognition. Full-

length protein antigens contain all possible human leukocyte antigen (HLA) epitopes

that can be processed and presented to T cells by antigen-presenting cells (APCs),

which is the greatest advantage of protein-based vaccines. However, similar to

peptide-based vaccines, protein-based vaccines must overcome the hurdle of low

immunogenicity, and as a result, adjuvant and/or fusion protein strategies have been used to enhance the potency of protein-based vaccines [66-68]. Because of the acyl moiety of bacterial lipoproteins, these agents are recognized as immunopotentiators by APCs, which that activate Toll-like receptor 2 (TLR2) signaling and elicit pro- inflammatory responses to trigger humoral and cytotoxic T cell responses

.

Bacteria express lipoproteins in the outer membrane for maintaining physiologic function. Lipoproteins were found to be a mitogenic for B cells because they contain the tripalmitoyl-S-glyceryl-cysteine moiety, for example outer surface protein A (OspA). OspA is the major outer membrane lipoprotein of Borrelia burgdorferi.

Recombinant lipidated OspA can stimulate B cell proliferation in a manner similar to native OspA [71]. Moreover, recombinant OspA provides co-stimulatory signals to induce human peripheral CD4

and CD8

T cell activation [72]. In 1998, the FDA approved the first lipoimmunogen-based vaccine for Lyme disease (LYMErix®), which was based on the OspA of B. burgdorferi [73]. Steere et al. designed a vaccine that consisted of full-length lipidated OspA, which was expressed by the E. coli strain AR58 and was mixed with aluminum hydroxide in phosphate-buffered saline.

Unfortunately, this vaccine was withdrawn due to complaints of autoimmune diseases even though no evidence of autoimmune complications was obtained [74].

The outer membrane lipoprotein of Pseudomonas aeruginosa (OprI) has been

used as a vector for the production of lipid-modified foreign proteins in an

Escherichia coli system [75]. Rau et al. demonstrated that OprI has adjuvanticity in

the context of a swine fever subunit vaccine in which humoral and cellular immunity

was enhanced without additional adjuvant [67]. However, the expression and

purification of recombinant lipoproteins are typically time-consuming tasks. Recently, we designed and engineered lipidated immunogens with TLR2 agonist activity by biologically linking antigens. The lipidated peptide sequence was identified from lipoprotein Ag473 of Neisseria meningitidis and expressed a large amount of the C43(DE3) strain of Escherichia coli [76]. The lipidated consensus E3 domain of the dengue viral envelope protein was found to contain unsaturated fatty acids in the lipid moiety [76]. The location of the unsaturated fatty acid in the lipid moiety was identified [77]. We further demonstrated that the recombinant lipoimmunogen stimulated TLR2-dependent activation of DCs but different cytokines compared with a synthetic tri-acyl lipopeptide [78]. ThereforeMoreover, the N-terminus of lipid moieties from the recombinant lipoimmunogen could be used as an adjuvant to improve the immunogenicity of protein/peptide-based vaccines or immunotherapies against cancers and/or infectious diseases [18].

Recombinant lipoimmunogens for therapeutic HPV vaccines

The same platform technology was applied to the development of a therapeutic

HPV vaccine. The mutated HPV16 E7 oncoprotein was used to produce a

recombinant lipidated E7 protein (rlipo-E7m). The endotoxin contaminations are

removed from rlipo-E7m by on column washing with 0.1% Triton X-100 / 50mM Tris

(pH 8.9) until the endotoxin levels are lower than 30 EU/mg. In contrast to the

synthetic lipopeptide, the recombinant lipoimmunogens are mixture that contained

different fatty acids on the same backbone as Pam3Cys. We demonstrated that rlipo-

E7m, but not non-lipidated HPV E7 (rE7m), elicited protective immunity against

tumors and robust E7-specific CTL responses following vaccination in mice. In an

animal study, a single dose of rlipo-E7m led to significant anti-tumor effects in both prophylactic and therapeutic models of mouse cervical cancer. Moreover, we found that the recombinant lipoprotein, which contained a triacylated N-terminus, induced a profile of cytokines and chemokines that was distinct from that induced by a synthetic triacylated lipopeptide (Pam3CSK4) even though both lipoimmunogenproteins elicited innate immunity via the activation of TLR2 signaling [78]. This discrepancy may indicate that the recombinant lipoprotein generated a different level of anti-tumor activity than the TLR2 agonist-conjugated peptide-based HPV vaccine. The therapeutic effects of the recombinant lipoimmunogen suggest that this technology represents a promising approach for the development of therapeutic vaccines against HPV-associated cancer.

TLR9 agonists enhance the anti-tumor activity of recombinant lipoprotein-based therapeutic HPV vaccines

Immunostimulator-conjugated peptides enhance antigen-specific CTL responses;

however, tumor-infiltrating immunosuppressive cells, tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs) may block their killing effects, resulting in so-called immune escape [79]. During tumor progression, cervical cancer cells can promote immunosuppressive cell differentiation. Cervical cancer cell lines release prostaglandin E2, and IL-6 promotes M2-like TAM differentiation [80].

To overcome the barrier of immunological tolerance induced by tumor cells,

successful cancer treatments must synchronize the blockade of immunosuppressive

networks, such as regulatory T cells (Tregs), MDSCs, and TAMs, and the induction

of tumor-specific CTLs [81-83]. Therapeutic approaches that use lipoimmunogens against HPV-associated cancers must enhance CTL responses and diminish tumor- infiltrating TAMs. Clodronate/liposome has been used to deplete macrophages to alter cervical cancer tumor growth [84,85]. In addition, immunization with a synthetic Pam2Cys peptide that contained HPV E7 CTL epitopes, combined with clodronate treatment, improved therapeutic outcomes in a TC-1 tumor model [54]. Furthermore, the synthetic Pam2Cys peptide immunization combined with a cyclooxygenase 2 (Cox-2) inhibitor (celecoxib) increased the survival rate of TC-1 tumor-bearing mice.

These results indicate that diminishing immunosuppressive cells might improve the therapeutic effect of lipoimmunogens in clinical trials.

In addition to eliminating tumor-associated immunosuppressive cells, several innate receptor agonists have been reported to modify tumor-infiltrating cell populations. TLR agonists are potential immunopotentiators for use in novel subunit vaccines. These agonists can elicit protective immunity against infectious diseases and/or cancer [86,87]. TLR2/TLR3 agonists and TLR3/TLR9 agonists have been demonstrated to synergistically activate dendritic cells and subsequently increase the number of activated T cells following vaccination [88]. Furthermore, Zhu et al.

demonstrated that the combination of TLR2, TLR3, and TLR9 agonists with an HIV peptide vaccine induced more effective CTL responses against viral challenge [89].

These results indicate that the combination of multiple TLR agonists results in high-

quality CTL responses. In addition to their synergistic effects on CTL responses, TLR

agonists regulate the immunosuppressive activity of myeloid-derived suppressive

cells (MDSCs) [90], tumor-associated macrophages (TAMs) [91,92] and regulatory T

cells (Tregs) [93,94]. These findings have inspired enthusiasm for the combination of multiple TLR agonists in the design of more effective therapeutic HPV vaccines.

Combination therapy with rlipo-E7m and other TLR agonists was used as an initial strategy to eradicate large, established solid tumors in a mouse model of HPV- associated cancer [95]. TLR agonists, including poly I:C (TLR3), imiquimod (TLR7), and CpG ODN 1688 (TLR9), were combined with rlipo-E7m, and their synergistic effects were examined using a BMDC activation assay. We found that the secretion of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-12p70, was substantially increased in plasmacytoid dendritic cells (pDCs) following stimulation with rlipo- E7m in combination with either CpG ODN or poly I:C. Interestingly, such synergy was not observed when rlipo-E7m was combined with imiquimod [95]. The use of CpG ODN as a cancer vaccine adjuvant was evaluated in clinical trials of vaccines against melanoma, non-small-cell lung cancer, and glioblastoma [96-99].

Encouraging clinical results demonstrated that CpG ODN boosted the immunogenicity of the vaccines and was associated with a superior safety profile when administered as a vaccine adjuvant.

Surprisingly, a single-dose subcutaneous injection of rlipo-E7m led to the

complete regression of large tumors when CpG ODN was coadministered 14 days

after tumor implantation in mice. The dramatic anti-tumor effects in on the tumor

infiltrate following the injection of rlipo-E7m/CpG may reflect the induction of long-

lasting and strong antigen-specific CTL responses and a reduction in

immunosuppressive cells (i.e., MDSCs, Tregs, and TAMs). The additive therapeutic

effects of the recombinant lipoimmunogen and CpG ODN were confirmed using

recombinant lipidated ovalbumin (rlipo-OVA) and CpG ODN. We found that rlipo- OVA induced higher levels of T cell proliferation in the presence of CpG ODN than rlipo-OVA alone in a co-culture of pDCs and OT-1

CD8

T cells (OT-1 is an OVA- specific TCR transgenic mouse). Interestingly, the additive therapeutic effects were not observed when a di-palmitoylated peptide was combined with CpG ODN (unpublished results). These results indicate that recombinant lipoimmunogens may have unique immunological activation profiles that are different from those of synthetic lipopeptides through the same TLR2 signaling pathway. Taken together, these findings suggest that a therapeutic formulation of recombinant lipidated E7m and a TLR9 agonist represents a feasible approach to potentially eradicating advanced tumors by eliciting substantial antigen-specific CTL responses against the cancer cell- expressing viral protein of HPV and by inhibiting immunosuppressive cells. However, the nuclease-resistant phosphorothioate CpG oligodeoxynucleotides (PS CpG) has been reported that induce some side effects and significant acute toxicity. Therefore, phosphodiester CpG ODNs (PO CpG) is biodegradable due to nuclease-sensitive and can be used as adjuvant to enhance immune responses in the future [100].

Conclusion

Lipoimmunogens are a promising strategy for the development of therapeutic

vaccines against HPV-associated cervical cancer. Synthetic palmitoylated peptides

have been demonstrated to elicit CTL responses against infectious diseases and

cancers. TLR2 agonist-conjugated peptides can promote high levels of CTL activation

without conventional adjuvants. Recently, recombinant lipoproteins have been

manufactured from bacterial expression systems, which is a novel strategy for the

development of self-adjuvanting molecular vaccines. In addition, recombinant

lipoproteins induce humoral immune responses that neutralize viruses, thereby

preventing persistent infection. Moreover, we found that the lipid structures of

synthetic palmitoylated peptides and lipoproteins varied as did the immune responses

they elicit [77,78]. We are currently investigating the CTL recruitment mechanisms of

a vaccine strategy that combines lipoimmunogens with CpG ODN. Recombinant

lipoproteins target DCs for antigen presentation and induce Th1-biased immune

responses for antitumor CTL activation.

Expert commentary

The concept of using synthetic lipopeptides in self-adjuvantinged vaccines is described in Table 1. The major application of these lipopeptides is in the development of vaccines against infectious diseases. Most clinical studies of lipopeptide vaccine candidates have demonstrated promising results. However, most of the neutralizing antibodies recognize conformational epitopes, but not linear epitopes, and that is difficult to mimic conformational epitopes using peptide, which may be a major obstacle to the widespread use of these vaccines. Antigen conformation is not necessary for inducing CTL responses; therefore, synthetic lipopeptides may be more suitable for therapeutic vaccine development. The following vaccine candidates have been examined in Phase I or II clinical trials: the Theradigm-HBV vaccine for HBV; the LIPO-4, LIPO-5 and LIPO-6 vaccines for HIV; and the PamLys-E7 peptide vaccine for HPV (Table 1). The main disadvantage of synthetic lipopeptides is HLA restriction. To overcome this limitation, recombinant lipoimmunogens may provide broader HLA-restricted epitopes. A recombinant lipoimmunogen product, LYMErix

, was approved by the FDA to prevent Lyme disease. The safety of this recombinant lipoimmunogen was not a major concern.

However, the product was withdrawn in 2002 because the sequence of the antigen

was similar to that of the human protein. The authors believe that recombinant

lipoimmunogens against cancer or infectious diseases will be tested in humans in the

near future.

Five-year view

Synthetic lipopeptides are easily utilized to produce GMP-grade materials. Clinical

studies of these lipopeptides will continue. Greater numbers of CTL epitopes of

different HLA and HPV types will be identified and incorporated into the synthesis of

lipopeptides. However, the long process of lipopeptide synthesis will be the major

challenge. Mixtures of multiple lipopeptides are a more feasible goal for the next 5

years. Moreover, GMP-grade recombinant lipoimmunogens for meningitis group B

have been produced and approved for a Phase I clinical trial in Taiwan. The authors

expect that the recombinant lipoimmunogen will be produced for clinical studies in

the next 5 years. Currently, recombinant lipoimmunogen technologies have been

applied to develop survivin-based and EBV protein-based reagents against cancer.

Key issues

1. Mono-palmitoylated lipopeptides cannot activate dendritic cells but are efficiently transported into the cytosol of APCs for priming CTL responses.

2. Self-adjuvantinged synthetic lipopeptides with TLR2 agonist activity can enhance the therapeutic effects of peptide-based vaccines.

3. Synthetic lipopeptides with TLR2 agonist activity regulate the cross-presentation of antigens and enhance the priming of CTL responses.

4. Immunization with recombinant lipoimmunogens induces Th1-biased immune responses and enhances CTL responses.

5. The lipid structures of synthetic palmitoylated peptides and recombinant lipoimmunogens vary, as do the immune responses they elicit.

6. Recombinant lipoimmunogens induce strong CTL responses and eliminate large tumors in the presence of a TLR9 agonist.

7. Recombinant lipoimmunogens combined with a TLR9 agonist efficiently reduce the number of systemic immunosuppressive cells and tumor-infiltrating

immunosuppressive cells, i.e., myeloid suppressive cells, TAMs and Tregs.

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Figure 1 Strategies for enhancing antigen presentation

(A) To target the MHC I presentation pathway, TAAs should be expressed as

intracellular antigens. Viral and DNA vectors encode the genes of TAAs that are

expressed in the cytosol and digested by the proteasome (Pr) to reveal CTL epitopes

in the peptides. These peptides can be transferred into the endoplasmic reticulum (ER)

by a transporter that is associated with antigen processing (TAP) for further trimming

and MHC I loading and then presented on the cell surface. Extracellular

protein/peptide antigens should be modified to target the cross-presentation pathway

for CTL cross-priming. Extracellular antigens are internalized by endocytosis into

early endosomes (EE) and either transferred to the cytosol for proteasome degradation

or led to late endosomes (LE) and lysosomes (Ly) for further digestion. These two

antigen-processing mechanisms can identify CTL epitopes in extracellular antigens

for MHC I loading and presenting. (B) Another approach is the up-regulation of co-

stimulatory molecule expression in DCs. TLR ligands, such as lipopeptides,

lipopolysaccharide (LPS), double-strand RNA or single-strand DNA (e.g., CpG), can

induce DC maturation. In addition, TLR ligands can facilitate antigen presentation via

the MHC I pathway or the cross-presentation pathway for CTL priming.

Figure 2 The cross-presentation mechanism of peptides and lipopeptides

Synthetic long peptides that contain a CTL epitope for eliciting CTL responses by DC

antigen processing and presentation. Non-, mono- and di- palmitoylated peptides have

distinctive internalization and antigen presentation pathways. (A) The non-

palmitoylated peptide is internalized by endocytosis into early endosomes (EE) and

cleaved by the proteasome. The digested peptide is transferred to the endoplasmic

reticulum (ER) for MHC I binding via a transporter that is associated with antigen

processing (TAP) and presented to the cell surface for CTL recognition. The non-

palmitoylated peptide is cross-presented by DCs via the TAP-dependent cross-

presentation pathway. (B) The mono-palmitoylated peptide can penetrate into the

cytosol by passive transportation for antigen processing by the proteasome. The

processed peptide is translocated to the ER for MHC I binding and presented for CTL

recognition. The mono-peptide can be presented by DCs via the TAP-dependent

antigen presentation pathway. (C) The di-palmitoylated peptide is endocytosed by

TLR2 into EEs. Rab7 regulates the maturation of late endosomes (LE) for the

processing of the di-palmitoylated peptide. The epitope of the di-palmitoylated

peptide is identified by lysosomes (Ly) for MHC I binding and presented for CTL

activation. The di-palmitoylated peptide can up-regulate Rab7 expression through

TLR2 signaling for cross-presentation via the vacuolar pathway.

Figure 3 Schemetic diagram of the (A) synthetic lipopeptide and (B) bacterial

lipoprotein.