Dendritic cells and vaccine design for sexually-transmitted diseases

Abstract

Dendritic cells (DCs) are major antigen presenting cells (APCs) that can initiate and control host immune responses toward either immunity or tolerance. These features of DCs, as immune orchestrators, are well characterized by their tissue localizations as well as by their subset-dependent functional specialties and plasticity. Thus, the level of protective immunity to invading microbial pathogens can be dependent on the subsets of DCs taking up microbial antigens and their functional plasticity in response to microbial products, host cellular components and the cytokine milieu in the microenvironment.

Vaccines are the most efficient and cost-effective preventive medicine against infectious diseases. However, major challenges still remain for the diseases caused by sexually-transmitted pathogens, including HIV, HPV, HSV and Chlamydia. We surmise that the establishment of protective immunity in the female genital mucosa, the major entry and transfer site of these pathogens, will bring significant benefit for the protection against sexually-transmitted diseases. Recent progresses made in DC biology suggest that vaccines designed to target proper DC subsets may permit us to establish protective immunity in the female genital mucosa against sexually-transmitted pathogens.

1. Critical features of DCs for host immunity

Dendritic cells are the major antigen presenting cells (APCs) that can induce and control host immune responses toward either immunity or tolerance [1–3]. DCs are also key immune mediators that link innate to adaptive immunity [4–7]. The roles of DCs in orchestrating host immune responses are not just limited to T cell-mediated cellular responses; DCs also play crucial roles in the induction and activation of antibody-mediated humoral responses in both healthy and diseases [8–12].

DCs localize throughout our bodies and, more importantly, position themselves where microbial pathogens invade. This feature of DCs defines them as the primary APCs. They capture the invading microbial pathogens at the frontline of infections, migrate to lymph nodes (LNs), and present antigens to lymphocytes to initiate antigen-specific adaptive immune responses [13, 14]. In the immune induction phase, DCs display two key features, functional specialty and plasticity, that can ultimately determine the outcomes of the host immune responses during and after microbial infections. The presence of phenotypically and functionally distinct subsets of DCs in local tissues, including skin [15–19] and different mucosae (reviewed in [20]), indicate that the pathways by which DCs orchestrate host immune responses are much more intricate than what we currently understand. Clearly, these pathways must be tightly regulated. Nonetheless, it seems that the functional specialization of different subsets of DCs is an important component for hosts to successfully defend against a variety of microbial pathogens, which invade through varying mechanisms.

The complexity of the mechanisms by which DCs orchestrate host immune responses is further extended by their ability to display functional plasticity. Microbial products and even certain cellular components are able to stimulate DCs through multiple receptors, particularly via pattern-recognition receptors (PRRs), such as toll-like receptors (TLRs) and C-type lectins [21–25]. Depending on the DC receptors that are ligated followed by the types and strength of intracellular signals delivered, DCs can be activated differently and thus can result in different outcomes of immune response. The plasticity of DCs also relies on elaborate signal codes that are generated by multiple stimuli simultaneously. Thus, the DC activation and maturation process is much more sophisticated than simply licensing DCs for the activation of lymphocytes. This particular process must also enable DCs to sense their environment and assume an activated phenotype that carefully instructs the qualitative nature of the immune responses. Taken together, DC subset-driven functional specialty and plasticity may allow DCs to cope with the challenges of their environment. These two features can eventually control and even dictate the magnitude and quality of the responses induced by vaccines and adjuvants. It is therefore fundamental to harness the functional specialties and plasticity of human DCs for the design of improved vaccines against microbial pathogens, including sexually-transmitted pathogens.

DCs also have unique and reciprocal interactions with innate immune cells. The interactions of DCs with natural killer (NK), NKT, and γδ T cells occur in the periphery and in the secondary lymphoid organs [6, 7, 26]. In the secondary lymphoid organs, the activation of NK cells is largely dependent on the interaction with DCs [27]. Activated NK cells display enhanced cytotoxicity and IFNγ secretion, which can further render DCs to induce type 1 immune responses [6]. Mature DCs also activate NKT cells to induce IFNγ and IL-4 secretion [28] and γδ T cells to induce IFNγ and TNFα secretion [29]. The roles of DCs in linking between innate and adaptive immune responses are further highlighted by the findings that activated NKT cells can kill tumor cells [26, 30], while CD40L expressed on the activated NKT cells induces the activation of DCs via CD40 [6].

2. DCs and immune tolerance

The ability of DCs to orchestrate host immune responses reaches beyond the elicitation and establishment of protective immunity against microbial pathogens. It also fulfills a key function for the induction as well as the maintenance of immune tolerance, which is crucial to protect the host from immune attacks driven by autoreactive lymphocytes (reviewed in [31, 32]). The first evidence that DCs were responsible for immune tolerance was provided by Kurts et al. [33] in a mouse model. They found that in the steady state, DCs present ovalbumin (OVA) peptides on major histocompatibility complex (MHC) class I molecules. OT I cells transferred to these mice proliferated in the draining LNs of kidney and pancreas but were eventually deleted. Other studies, using direct DC targeting of antigen, suggested that DCs are the APCs that induce immune tolerance for self-antigens in vivo [31, 32]. Targeting non-self antigens through DEC-205 also resulted in tolerance by the deletion of antigen-specific CD4⁺ T cells, whereas combined administration of DC-targeted antigen with an agonistic anti-CD40 antibody led to prolonged T cell activation [2, 34]. Furthermore, injection of mice with dying syngeneic TAP^−/− splenocytes loaded with OVA led to presentation of cell-associated OVA by the CD8α⁺ DCs, followed by deletion of OVA-specific CD8⁺ T cells [35]. In humans, Dhodapkar et al. [36, 37] first demonstrated that the injection of immature DCs pulsed with influenza matrix peptide and keyhole limpet hemocyanin (KLH) in healthy individuals resulted in decreased matrix-peptide-specific IFN-γ-producing CD8⁺ T cells. Interestingly, they also found that those healthy subjects had increased numbers of the same antigen-specific IL-10-producing T cells. Taken together, these observations suggest that the outcome of antigen presentation by DCs in the steady state may be systemic antigen-specific tolerance. Therefore, immature DCs residing in peripheral tissues can act as tolerogenic APCs. They can capture a broad spectrum of antigens, including autoantigens, through different mechanisms [14]. Unless there is a proper activation signal, they become tolerogenic [34, 38, 39] .

However, the nature of tolerogenic DCs needs to be further investigated. For example, a constitutive presentation of H⁺/K⁺ ATPase by DCs does not induce autoimmunity nor ATPase-specific T cell tolerance [40]. Other immunoregulatory mechanisms might also be involved in the induction and maintenance of antigen-specific immune tolerance. Recent studies have similarly shown that immune tolerance can be induced by DCs matured with endogenous non-inflammatory signals [41, 42]. We have additionally reported that antigens targeted to DCs via one of the PRRs, DC-asialoglycoprotein receptor (DC-ASGPR) [43], can induce antigen-specific IL-10-producing regulatory T cells both in human in vitro and non-human primate in vivo. More importantly, this was applied to both foreign and self antigens as well as to both naïve and memory T cell responses. DCs express a variety of PRRs (reviewed in [21–25]) that can specifically recognize different types of microbial as well as host-cell-driven endogenous stimuli. Therefore, DCs must be able to discriminate between self and non-self, and they must enable the immune system to mount immunity to pathogens while silencing auto-reactive lymphocytes [38, 44].

3. Critical features of DC subsets for the design of vaccines against STDs

After Ralph Steinman and his colleagues discovered DCs in the early 1970s [45, 46], our understanding of the role of DCs as immune orchestrator and their application to human medicines has been well progressed. DCs are now divided into two major subsets, myeloid DCs (mDCs) discovered by Ralph Steinman’s group and plasmacytoid DCs (pDCs) discovered by Yong-Jun Liu’s group in 1997 [47]. Both mDCs and pDCs are found in blood as well as in tissues. One of the major characteristics of pDCs is the secretion of high levels of type 1 IFN in response to viral infection [48]. pDCs also play an important role in innate immunity [49]. Although the contribution of pDCs in the direction of T cell priming in vivo remains to be further characterized, pDCs are also able to present antigens via MHC I and II [50–53]. pDCs have also been implicated in the induction of tolerance [54, 55].

The presence of phenotypically and functionally distinct mDC subsets in different tissues (reviewed in [56]) and their plasticity recapitulate the value of DCs as the primary targets for the design of effective vaccines. DC subsets in blood, lymphoid organs, and other mucosae have been extensively reviewed [57–68]. Below, we review the subsets of DCs and their critical features that need to be considered for the design of effective vaccines against sexually-transmitted pathogens. Although we propose to design vaccines to target DCs in the female genital tract, particularly in the vaginal mucosa, such vaccines might also be administered via non-mucosal routes or through a combination of mucosal (intravaginal: IVAG) (reviewed in [69]) and non-mucosal routes. In this review, therefore, we discuss the subsets of skin DCs and the DCs in the female genital tract, especially in the vaginal mucosa. The DC network in the human vagina has not been well understood. However, data from recent studies in animals [70] and humans [71, 72] have shown that DCs in the vaginal mucosa can be the immune initiators and controllers in the vaginal mucosa and female genital tracts.

3.1. Skin and skin DC subsets

3.1.1. Mammalian skin

Skin and mucosae are the outermost anatomical barriers against pathogens as well as against damages from internal and external stresses. Mammalian skin is composed of the epidermis and dermis. The outermost layer, the epidermis, is mainly composed of proliferating basal cells and differentiated supra keratinocytes. It also contains Merkel cells and melanocytes as well as Langerhans cells (LCs). The proliferating basement membrane between the epidermis and dermis can control the traffic of cells as well as biomolecules, including cytokines and growth factors, between the two major compartments. The dermis is composed of connective tissues that cushion the body from stress and strain. It also contains the hair follicles, sweat glands, sebaceous glands, apocrine glands, lymphatic vessels and blood vessels as well as dermal DCs, lymphocytes, and other immune cells.

3.1.2. Skin DC subsets

In human skin, there are three major subsets of mDCs: LCs are in the epidermis while CD14⁺ and CD1a⁺ DCs are in the dermis [15]. In vitro studies show that subsets of skin-resident mDCs can display specialized functions to induce and activate T and B cell responses. LCs are more efficient than CD14⁺ dermal DCs at cross-presenting antigens to CD8⁺ T cells. Skin LCs can also induce CD4⁺ T cells to secrete Th2-type cytokines. Similar observations were made in a mouse in vivo study. Upon delivery of antigens to LC-rich epidermis, Th2-type T cell responses were preferentially induced [16]. Interestingly, transcutaneous immunization of antigens to mice resulted in potent CD4⁺ and CD8⁺ T cell-mediated protective immunity to viruses [73, 74]. In contrast to LCs, CD14⁺ dermal DCs can polarize naïve CD4⁺ T cells into follicular helper T cells (Tfh) and can thus prime humoral immunity [17]. Skin LCs are also known to induce Th17 responses [18, 75]. In addition, skin LCs can efficiently activate regulatory T cells [76], as well as Th22-type CD4⁺ T cell responses [19]. Nonetheless, these immunological functions of human skin DC subsets for the induction and activation of T and B cell responses are yet to be established in vivo.

In mice, LCs can present exogenous antigens on MHC I and MHC II both in vitro and in vivo [77–79]. In contrast, a study using herpes simplex virus (HSV) has questioned the contribution of LCs to the induction of antigen-specific responses in vivo. In this study [80], HSV-specific T cell responses were not due to the LCs, but to CD8α⁺ DCs. Furthermore, another study also showed that dermal CD103⁺ DCs, but not dermal CD11b⁺ DCs or LCs, present antigens to naïve CD8⁺ T cells [81]. The roles of CD103⁺ DCs in cross-priming antigen-specific CD8⁺ T cells have been further confirmed by other studies [82–84]. It still remains to be determined whether these differences with regard to the function of LCs between mice and humans derive from the differences in their immune systems. All DCs are capable of presenting viral antigens to CD4⁺ T cells [85].

3.2. Vaginal mucosa and DC subsets in the vagina

3.2.1. Female genital tract and vaginal mucosa

The female genital tract is comprised of the upper (oviducts, ovaries, uterus, and endocervix) and lower (ectocervix and vagina) reproductive tract [20]. The upper tract is covered by a simple columnar epithelium (type I mucosa) [86] that is hormonally regulated for fertilization and fetal development. The columnar epithelium is characterized by the presence of tight junctions between cells, making it impermeable to entry of large molecules, including microbial pathogens. In contrast, the lower reproductive tract is lined with more than 25 layers of stratified squamous epithelial cells (type II mucosa) [69, 87–89]. The epithelium in the lower tract, although not impermeable, is robust and provides a substantial physical barrier. Only type I mucosal epithelium expresses polymeric immunoglobulin (Ig) receptors, which can transfer dimeric IgA to the lumen [69, 90, 91], with IgA being the abundant isotype in the type I mucosa. In contrast, antigen-specific antibodies in the vaginal mucosa are dominated by IgG [88]. The epithelium as a physical barrier and the interaction between the epithelium and sexually transmitted pathogens have been recently reviewed [92, 93].

The lower female genital tract, particularly the vaginal mucosa, is constantly exposed to foreign antigens and is a unique microenvironment that must control unwanted types of immune responses [69, 94–96]. Meanwhile, the vaginal immune system is also capable of eliciting mucosal immune responses in the vagina [20, 71, 97–105]. Furthermore, intravaginal (IVAG) administration of vaccines can elicit certain levels of immune responses in the vagina in animal models [97–100] as well as in humans [71, 101–105].

Nonetheless, the protective immunity induced in the vaginal mucosa by the current mucosal vaccine models needs to be enhanced [69]. As part of the need for new approaches, a new clinical trial of HIV trimeric gp140 protein CN54 (Infectious Disease Research Institute: IDRI) is currently underway and data from this trial will be available in 2013. In particular, the establishment of cellular immunity, which is a critical immune arm against many sexually-transmitted viruses, has been one of the major challenges for the design of vaccines against sexually-transmitted diseases. In this regard, we envision that safe vaccines that can mount potent cellular as well as humoral immunity in the female genital tract, including the vagina, can be developed in the near future. However, such vaccines may need to be designed to target the right antigens to the right subsets of DCs in the vaginal mucosa. Selection of optimal adjuvants that can further promote the vaccine-targeted DC subset-induced immune responses may also determine the efficacy of new vaccines. Thus, understanding the immunology of the human vagina, especially for the subsets of DCs and their functional specialties and plasticity, will be fundamental for the design of such vaccines.

3.2.2. Vaginal DC subsets

Early studies in animals showed that the type II vaginal mucosa, which is covered with stratified squamous epithelium [69, 87, 88], shares several common features with the skin. LCs are found in the epithelium and CD11c⁺ DCs in the lamina propria (LP) [88, 94]. There are at least four populations of LCs in the epithelium that have been identified: I-A⁺/F4/80⁺, I-A⁺/F4/80^–, I-A⁻/CD205⁺, and I-A⁺/CD205⁻ populations by immunohistochemistry [106] and CD11b⁺ F4/80^hi, CD11b⁺ F4/80^int, and CD11b⁻ F4/80⁻ populations by flow cytometry [107]. However, it is not known whether these populations have specific functions in the immune responses of the vaginal mucosa. DC-SIGN⁺ CCR5⁺ LP-DCs were also reported in animals [108, 109]. In mice, both lymphoid DCs and tissue-resident DCs are known to prime T cell responses in mice infected with HSV-1 [110]. Zhao et al. [70] also reported that vaginal LP-DCs, but not LCs, are capable of inducing protective Th1-type responses to HSV-2 infection in mice. However, during infection or inflammation, both monocyte-derived DCs [107] and pDCs [111] are recruited from peripheral blood to the vaginal mucosa.

In humans, the presence of LCs in the vaginal epithelium is known [112, 113], but no further information is yet available. Moreover, the immunological functions of DCs localized in the human vagina remain unknown. Below, we review the subsets of DCs known to localize in the human vagina, expression of PRRs, and DC functional plasticity, based upon our recent study [114].

Human vaginal mucosa contains four major subsets of myeloid-originated APCs: CD207⁺ LCs in the epithelium, CD1c⁺CD14⁻ DCs (CD14⁻ LP-DCs), CD1c⁺CD14⁺ (CD14⁺ LP-DCs), and CD1c⁻CD14⁺ macrophages (Mϕ) in the LP (Fig. 1). In a steady state, the frequencies of pDCs, B cells, and BDCA3⁺ cells in the human vagina are low. These four major APC subsets display shared and distinct surface phenotypes. LCs express both CD1a and E-cadherin. LCs, as well as the CD14⁻ LP-DCs and CD14⁺ LP-DCs, express both CD86 and CD83. CD1c⁻CD14⁺ cells express CD86, but not CD83. Mϕ express CD163 and CCR2, a homing receptor for monocytes and Mϕ [115]. CCR5 and CXCR4, co-receptors for HIV, are expressed on all four subsets of APCs [116]. Interestingly, the vaginal LP-DCs and Mϕ express increased levels of both CCR5 and CXCR4 compared to LCs. Both LCs and LP-DCs exhibit similar levels of CCR6, which is known to be expressed on intestinal DCs [117]. β7-integrin is detected on LCs and LP-DCs, but not on Mϕ. Both CCR4 and CX3CR1 are similarly expressed on the four subsets of APCs in the vagina, while CCR7 and CD103 are not detected on the surface of the vaginal APC subsets. Taken together, these characteristics of the four major subsets of vaginal APCs suggest that each subset of the APCs might possess both common and distinct functions in directing the immune responses in the vagina.

Figure 1. Human vaginal mucosa harbors four major populations of antigen-presenting cells that display shared and distinct functions at inducing T cell responses.

Four subsets of myeloid-derived APCs are found in human vaginal mucosa: LCs in the epithelium and CD14⁻ LP-DCs, CD14⁺ LP-DCs and macrophages (MØ) in the submucosa. Each population displays shared and distinct phenotype and functions at inducing naive T cells responses.

In support of the expression levels of co-stimulatory molecules, the vaginal DCs, including LCs, are able to induce greater allogeneic CD4⁺ and CD8⁺ T cell proliferation than Mϕ. All four subsets of the vaginal APCs induce similar percentages of IFNγ⁺CD4⁺ T cells, but LCs and CD1c⁺ LP-DCs are more potent than CD14⁺ LP-DCs and Mϕ at inducing Th2-type CD4⁺ and CD8⁺ T cell responses. However, the vaginal APC subsets are not able to induce significant levels of Th17 responses, although Th17 cells contribute to the protective immunity against mucocutaneous candidiasis [118–120] and skin DCs are known to be potent Th17 inducers [18]. Furthermore, none of the vaginal APC subsets display functional specialty to polarizing T cell differentiation into Tfh. In contrast, CD14⁺ skin dermal-DCs are potent inducers of IL-21⁺CD4⁺ T cells [17]. Furthermore, vaginal LCs and CD14⁻ LP-DCs induce similar levels of naïve CD4⁺ T cell proliferation, whereas skin LCs are superior to CD1a⁺ dermal DCs at inducing naïve CD4⁺ T cell proliferation [17, 121]. These features of the vaginal APC subsets clearly distinguish them from the subsets of APCs in the skin. The subset-dependent functional plasticity of the human vaginal APCs can be further promoted by activating DCs through different PRRs. Vaginal DCs express increased levels of PRRs, including MDA-5 [122] and TLR7, which play important roles in anti-viral immune responses. Mϕ express increased levels of TLRs 1, 2, 4, and 6, which allow them to recognize bacterial products. The roles of IL-22 in adaptive immune responses have not been well understood. However, the vaginal LCs and CD14⁻ LP-DCs are able to induce Th22 responses that are further promoted by R848, a ligand for TLRs 7 and 8. Taken together, data from our recent study [114] illustrate that each subset of the DCs and Mϕ in the human vagina can display common as well as distinct functional specialties and plasticity. These features are critical for the design of effective vaccines against sexually-transmitted diseases, although a series of studies to further understand the immunology of the human vagina needs to be done.

4. DC-targeting vaccine for STDs

The pivotal roles of DCs in immunity make them an attractive target for the manipulation of host immune responses [57, 123]. To date, attempts to leverage the use of DCs in vaccination have largely involved cell-based approaches, particularly against cancers. For instance, the in vitro generation of DCs, loading DCs with antigens and the injection of the antigen-loaded DCs to patients, have been explored by several groups (reviewed in [124]). However, this approach has limitations in several aspects, including the preparation of vaccines and antigens as well as costs. In addition, clinical efficacy of such vaccines still needs to be improved. Therefore, the rationale to the design of in vivo DC-targeting vaccines has been well accepted in the fields of immunology and vaccines. A study from a decade ago showed that a minute amount of protein antigens targeted to in vivo DCs via the C-type lectin DEC-205 together with anti-CD40 agonistic antibody results in antigen-specific CD8⁺ T cell responses in mice [125]. In the absence of adjuvant, however, targeting protein antigens to in vivo DCs via DEC205 resulted in antigen-specific immune tolerance [34, 35]. In the last decade, studies have shown that the DC-targeting vaccine strategy is an efficient way to mount both cellular and humoral immunity [125–127]. The efficiency of antigen presentation on MHC class I and II molecules was increased approximately 100-fold by targeting protein antigens to the DC surface receptors compared to protein antigen without targeting [125, 128, 129]. In addition to DEC205, studies have shown that protein antigens targeted to DCs via a number of other cell surface receptors, such as DC-SIGN [130], CD1b [131], LOX-1 [132], and the mannose receptor (MR) [133–136] are able to elicit antigen-specific immune responses in vitro and in vivo. Table 1 summarizes receptors that are currently being investigated for the design of antibody-based in vivo DC-targeting vaccines. Human trials with prototypes of DC-targeting vaccines composed of anti-DEC205 and anti-MR antibodies are currently ongoing.

Table 1.

Current studies on the antibody-based in vivo DC targeting vaccines.

Targeting receptors	Experimental models	Antigens	Adjuvants	Immune responses	References
Mannose Receptor (MR)	Mouse: Human MR transgenic mouse	OVA	CpG	Abs, CD4+ and CD8+ T cells	[153, 154]
	Human in vitro	Chorionic gonadotropin beta subunit	CD40 ligand	CD4+ and CD8+ T cells	[155]
		NY-ESO-1		CD4+ and CD8+ T cells	[156]
		pmel17		CD4+ and CD8+ T cells	[136]
DEC205	Mouse	OVA	Agonistic anti-CD40 mAb	CD4+ and CD8+ T cells	[34, 35, 125]
		HIVgag	Poly IC and agonistic anti-CD40 mAb	CD4+ T cells	[128]
	Human in vitro	NY-ESO-1		CD4+ and CD8+ T cells	[156]
		HIVgag-p24		CD4+ and CD8+ T cells	[157]
DC-SIGN	Mouse	OVA	LPS	CD4+ and CD8 T cells	[158]
	Human in vitro	KLH		CD4+ and CD8+ T cells	[159]
LOX-1	Mouse cancer model	OVA		CD8+ T cells	[132]
	Human in vitro	Influenza HA1 and PSA		CD4+ T cells	[43]
DCIR	Human in vitro	Influenza M1, MART-1, and HIVgag	TLR7/8	CD8+ T cells	[138]
Langerin	Mouse	HIV Gag-p24	Agonistic anti-CD40	CD8+ T cells	[160]
Dectin-1	Mouse	OVA	TLR3 ligand	CD4+ and CD8+ T cells	[161]
	Human in vitro	MART-1 peptide and Influenza M1		CD8+ T cells	[137]
		Tumor cells and CMV	TLR2 and TLR7 ligands	CD4+ and CD8+ T cells	[162]
Clec9A	Mouse	OVA	CpG, Poly IC, and LPS	Abs, Follicular CD4+, CD8+ T cells	[126]
CD40	Human in vitro	NY-ESO peptide Influenza M1 and CMV peptides		CD8+ T cells	[139] [139]

In principle, we can apply the current concept that has been developed and tested by others and by us for the development of DC targeting vaccines against sexually-transmitted pathogens and diseases, such as cervical cancer. To design optimal DC-targeting vaccines that can elicit potent and protective cellular and humoral immunity in the female genital tract, however, there are still a number of questions that need to be addressed. First, the receptor targeted by vaccines should be preferentially expressed on DCs, particularly on the surface of the selected DC subsets. This allows for further induction of the desired types of immune responses without triggering unwanted types of responses. Receptors targeted by the current experimental vaccine models are summarized in Table 1. Data from our studies show that C-type lectins such as LOX-1 [43], Dectin-1 [137], DC-ASGPR [43], and DC-SIGN (not shown) are mainly expressed on dermal DCs in the human skin, while DCIR is expressed on both LCs and dermal DCs [138]. However, the expression of such candidate receptors on the surface of human vaginal APCs are yet to be fully investigated. Our recent data show that cells expressing DC-SIGN, LOX-1, and DCIR are mainly localized in the LP of the human vaginal mucosa (Fig. 2). Both DEC205 and Langerin are mainly expressed on the cells in the epithelium. Data in Fig. 2 also suggests that cells expressing DC-SIGN, LOX-1, and DCIR are not necessarily the same. It is assumed that subsets of DCs and macrophages in the human vaginal LP express shared as well as distinct C-type lectins. Taken together, these data suggest that vaccines targeting selected subsets of the vaginal APCs could be designed. However, the expression of DC surface receptors on the subsets of the vaginal APCs is yet to be more extensively investigated.

Figure 2. DC receptors expressed in human vagina mucosa.

Immunohistochemistry staining of frozen tissue sections with anti-DC-SIGN (clone 15C4, in house), anti-LOX-1 (clone 15C4, in house), anti-Langerin (clone 15E2, in house), anti-DEC205 (clone MG38, eBioscience), anti-DCIR (clone 9E8, in house) antibodies or isotype controls. Digital images were taken using an Olympus BX60 with a UPlanFl 10×/0.30 Ph1 objective, a Nikon Digital Camera DXM 1200C camera and Nikon Elements software (Nikon). (×10, bar is 100µm)

Second, antigens targeted to the receptor should be properly internalized, processed and presented in both MHC classes I and II. For instance, CD40 and MR targeted antibody conjugates to early endosomes, whereas DEC205 targeted antigen primarily to late compartments [139]. The receptor least efficient at internalization, CD40, was the most efficient at cross presentation. This did not reflect DC activation by CD40, but rather its relatively poor uptake or intra-endosomal degradation compared with MR or DEC205. These features of individual candidate receptors are crucial elements that certainly need to be considered for the development of optimal DC-targeting vaccines.

Third, selection of optimal adjuvants that can further promote the functional specialties of the targeted DC subsets will be another critical factor for the design of DC-targeting vaccines. Thus, strategies aimed at inducing immunity require more than just targeting of antigen to the selected subsets of APCs. In addition to the types of adjuvants, timing of antigen delivery and maturation stimulus are also crucial for eliciting strong immune responses that lead to the establishment of protective immunity. Data from the first phase 1 trials assessing in vivo DC-targeting vaccines composed of anti-MR antibody show that stronger immune responses and longer durations of immune responses are observed when the vaccines are co-administered locally with TLR agonists [140]. For safety reasons, adjuvants will not be given systemically. Blander and Medzhitov have also reported that the efficacy of antigen presentation from phagocytosed cargo largely rely on the presence of TLR ligands within the same cargo [141]. This could be due to the governing of MHC class II-restricted antigen presentation by TLR-induced signals occurring in the phagosomes as antigens are delivered. Another study also reported that conjugating antigens with TLR agonists enhanced the magnitude and quality of Th1-type CD4⁺ T cells and CD8⁺ T cell responses in nonhuman primates [142]. However, if engagement of several DC surface receptors by anti-DC surface receptors alone triggers immunostimulatory signaling pathways, this could preclude the need of adjuvant.

Fourth, vaccines need to contain proper antigenic components that can bring protective immune responses. Thus, appropriate antigens for targeted microbial pathogens must be carefully chosen. Antigens of sexually-transmitted pathogens and the types of immune responses, humoral and cellular, to the pathogens are summarized in Table 2. These protein antigens can be incorporated into the vaccine prototype, but need to be further tested.

Table 2.

Protein antigens of sexually-transmitted microorganisms

Pathogen	Protein Ags	Type of immune responses		References
		T cell responses	B cell responses
HIV	gp41, gp120, gp160, gag, pol, nef	yes	yes	[163–165]
HPV	E6, E7, L1	yes	yes	[166–170]
HSV-2	gpB, gpD and gpG	yes	yes	[171–174]
Chlamydia trachomatis	CprA, OMP2, MOMP, CT144, CT823, PorB, PmpD, pgp3	yes	yes	[175–179]
Candida albicans	Als1/Als3, MP65, Fba, Met6, Hwp1,	yes		[180–182]
Neisseria gonorrhoeae	Opa		yes	[183, 184]
Treponema pallidum	Trp, Tp92	yes	yes	[185–187]
HCV	HCV-core, NS3, NS4, NS5, lipopeptides	yes	yes	[188, 189]

Finally, the efficiency with which the antigen is delivered to the DCs is largely dependent on the choice of immunization routes. In general, DCs originating from a specific tissue have the ability to instruct lymphocytes to home back to that tissue [143]. Different DC subsets in the same tissues might provide even more detailed instructions. Thus, the rationale for the IVAG administration of DC targeting vaccines can be well accepted. Furthermore, such mucosal vaccines are expected to elicit strong mucosal immune responses followed by the establishment of protective immunity in the female genital tract. However, it is important to keep in mind that the microenvironment in the lower female genital tract is unique in terms of the types of biological factors that could influence the outcomes of vaccine-induced immune responses. Female sex hormones [92, 144, 145], use of hormonal contraception [146, 147] and depo-medroxyprogesterone as well as pregnancy [148], could certainly alter the functions of APCs in the vagina. Epithelial cells in the vaginal mucosa express a variety of TLRs [149–151], and so the immune responses elicited by IVAG administration of vaccines with free TLR ligands could be influenced by the epithelial cells [92, 93, 145, 152]. To avoid or minimize such effects, adjuvants may need to be conjugated to the vaccines. However, it is still important to ensure that the adjuvants do not dampen the types of immune responses elicited by the subsets of DCs targeted by the vaccines. In addition, the presence of soluble factors (reviewed in [87]), including interleukin (IL)-1α, IL-6, and transforming growth factor-β, secreted from epithelial cells also need to be considered.

In conclusion, the DC targeting protein vaccines described in this review are a potential new vaccine platform. Both antigens and adjuvants can be carried by antibodies specific for the selected vaginal DC subsets. There are still several scientific and practical questions, such as vaccine formulation and delivery, that need to be better understood. However, it is certainly possible that vaccines designed based on our current knowledge on the DCs could establish protective immunity in the female genital tract that could prevent sexually-transmitted microbial infections. These vaccines can also be considered in males, too.