Background
There is currently no approved vaccine against HIV despite considerable efforts in vaccine research and clinical trial. Given the important role of mucosal surface as the port of entry for HIV, effective immunization strategies should be those that elicit significant immune responses at mucosal sites. While intramuscular(i.m) injection has been proven useful for stimulating systemic immune responses, it induces poor responses at mucosal surfaces [
1]. Vaccine delivery via mucosal route provides potential opportunities for induction of effective mucosal responses. When administered in combination with systemic vaccination, mucosal immunization, including oral [
2], intranasal (i.n.) [
3,
4] as well as intravaginal (IV) routes [
5,
6], has been proven highly potent in inducing effective mucosal immune responses.
Intensive studies have been conducted to develop and investigate potent mucosal adjuvants for improved vaccine potency. Several stimulatory molecules are suggested as effective adjuvants to enhance mucosal immune response, these including cholera toxin,
Escherichia coli heat-labile toxin, CpG motif, and cytokines [
7‐
9]. However, to date no licensed immunopotentiating mucosal adjuvant is available. Chemokines are a group of small proteins that play important roles in innate and acquired immunity by regulating inflammation, leukocyte trafficking, and immune cell differentiation [
10‐
12]. Chemokine (C-C motif) Ligand 20 (CCL20), also known as Macrophage Inflammatory Protein (MIP)-3-alpha or Liver Activation Regulated Chemokine (LARC), has been shown to be expressed constitutively in variety of normal human mucosa-associated tissues, especially in the mucosal epithelial cells. CCL20 plays an important role in mucosal homeostasis through attraction of immune cells including DC, T and B-lymphocytes [
13,
14]. Meanwhile, CCL20 itself is regulated. For instance, the presence of inflammatory mediators promotes the up-regulation of CCL20 in mucosal epithelial cells [
15,
16]; other studies have also demonstrated that the colonic epithelial cells from patients with inflammatory bowel disease produce higher level of CCL20 [
17,
18].
Neutralization of CCL20 expression by its monoclonal antibody has been shown to reduce T cell recruitment [
19]. This finding demonstrates that CCL20 contributes to the enhanced recruitment of its potential target cells, which are known to home preferentially to these sites of continuous antigen-challenge, such as the epithelial mucosal surface [
20]. Given the important physiological roles of CCL20 in immune regulation, leukocyte trafficking and immune cell differentiation, we set out to find out whether such biological functions could be employed to enhance mucosal immune response, especially these against HIV immunogen. In this study, CCL20 has been incorporated into HIV Env Virus-Like Particles (VLPs) and its immunomodulatory role was investigated.
Discussion
Mucosal transmission of HIV infection accounts for as high as 80% of AIDS [
28]. It is generally believed that the induction of effective immune responses at mucosal portals of viral entry offers the best protection against HIV [
29]. To date, an HIV vaccine with fully protective efficacy is not yet available. An effective HIV vaccine should not only induce systemic immune response to clear disseminated viruses, but also promote mucosal immune responses to block transmission. Our previous studies have shown that incorporation of cytokines as adjuvants promotes enhanced mucosal and systemic responses with improved quality [
30]. The important roles that cytokines play in modulating immune responses induced by various vaccines have also been established [
31]. Given the important roles that CCL20 and its receptor CCR6 play in leukocyte maturation, migration as well as mucosal immunity, we set out to investigate the potential of this chemokine as a molecular adjuvant for HIV vaccine for improved mucosal and systemic responses.
In the present study, engineered membrane-bound CCL20 was co-incorporated with HIV Env into VLPs and the resulted cVLPs were tested for their efficacy in promoting enhanced immune responses. Data from Fig.
2 strongly indicated that the co-incorporation of CCL20 into VLPs arguments both serum as well as mucosal immune responses as reflected by significantly improved IgG and IgA titers in serum as well as vaginal samples from i.n and IV boost groups. IgA is an important type of mucosal antibody involved in the first line of defense of pathogens and capable of inhibiting virus transcytosis through epithelial layers and has potent neutralizing activity [
15]. Therefore, a vaccine capable of eliciting IgA response could be beneficial by contributing to the containment of HIV infection.
It is well established that T cell responses are crucial for clearing viral-infected cells and controlling viral replication. T cell responses are beneficial to host immune defense against HIV infection [
32,
33], and CD4
+ T cell responses are critical for humoral responses. We evaluated the T cell responses (Ag-specific Th1- and Th2 associated cytokine induction) induced by various VLPs. Results in Fig.
3 demonstrated that incorporated CCL20 enhanced both Th1 and Th2 responses in mice when the cVLPs were boosted by i.n. or IV routes.
To further confirm the adjuvant effect of CCL20, the level of Env-specific IgA ASCs was evaluated by ELISPOT. Our data (Fig.
4a) suggested that cVLPs induced elevated level of Env-specific IgA ASCs as compared to standard VLPs. To verify the contribution of CCL20 to the elevated level of IgA ASCs and homing of Ag-specific IgA ASCs into small intestine in mice immunized with CCL20, CCL20 was neutralized by its monoclonal antibodies. The level of Env specific IgA ASCs in the lamina propria was reduced (Fig.
4b), indicating that the homing of Env specific IgA ASCs was partially blocked by anti-CCL20 in small intestines. The impaired IgA ASCs level implied that CCL20 may play a role in improving IgA responses and the partially blockage by anti-CCL20 antibodies can be explained as the following. The chemokine and their receptors contribute to accumulation of specific leukocytes in various tissues [
34], and it is well established that chemokine and tissue-specific adhesion molecules are involved in the migration of Ab-secreting cells. It is possible that some Env specific IgA ASCs are still capable of entering into intestinal tissues by responding to other chemotactic factors, such as CCL25, CCL28 and their corresponding receptors CCR9 and CCR10, which enables plasma B cells to migrate to mucosal tissues [
35]. Mucosa-associated Epithelial Chemokine (MEC/CCL28), a well-known ligand for CCR10, is commonly produced by epithelial cells at several mucosal sites and attracts IgA- but not IgG or IgM-producing ASCs into the intestines, lungs, and oral cavity [
36]. Others reported that CXCR4 and its ligand CXCL12 are involved in the translocation of plasma cells within the splenic and lymph node as well as in their homing to the bone marrow [
12]. Additionally, down-regulation of CCR7 and CXCR5 will allow the B cells to migrate to efferent lymphatic vessels [
37]. Further, CCL25 plays essential roles in intestinal homing of IgA ASCs [
38]. Taken together, it is possible that the residual IgA ASCs were detectable due to other chemokine activities which can exert influence on the migration of lymphocytes.
Based on the above results, we concluded that immunization with cVLPs containing membrane anchored CCL20 induced enhanced Env-specific serum and mucosal immune responses, cytokine secretion, as well as IgA
+ plasma cell production. It is noteworthy that, in contract to the VLP-incorporated CCL20, the soluble CCL20 has no adjuvant effect by i.n. nor IV immunization when mixed with HIV standard VLPs based on data shown in Figs.
2,
3 and
4. The different delivery approaches of CCL20 may contribute to the difference in association between CCL20 and antigen and therefore the adjuvant function of CCL20. In cVLPs, co-incorporated CCL20 is more likely to be co-presented with Env and accessed by APCs due to the established advantage of VLPs as efficient vaccine platform [
39,
40]. CCL20 can bind to surface CCR6 on APC or other lymphocytes and thus effectively facilitate VLP uptake and antigen processing and presentation. In contrast, soluble CCL20 may be poorly associated with Env and may not interact with or recruit APCs as efficiently as its VLP-incorporated counterpart.
Another important question we asked in this study is whether immunization boost by i.n. or IV route is capable of inducing enhanced mucosal immune responses. The Gut Associated Lymphoid Tissue (GALT) contains the majority of T cells, and many studies have shown that GALT is the preferential target for HIV replication during infection [
13,
14]. Therefore, mucosal vaccine holds great promise for effective HIV prophylactic. Our data suggest that i.n. or IV boost indeed induced highly augmented levels of IgG and IgA in vaginal wash when mice were immunized with cVLPs. In contrast, i.m. administration and boost of HIV cVLPs induced mainly systemic rather than mucosal immunity (Fig.
2). Further, i.n. or IV boost also elevated the level of Env-specific IgA ASCs in the intestine lamina propria as well as cytokine secretion (Figs.
3 and
4). It is intriguing to check out if i.n. boost, as a non-invasive route, has any different effect on mucosal immune responses compared to IV. As suggested by our data, no significant advantage of i.n. over IV boost. The primary function of the mucosal immune system is to protect the host from invading pathogens. It is well established that immunization route is critical in determining the nature of induced immune responses. Our data generally support the notion that i.m. route may favor the induction of systemic response, whereas i.n. or IV route might promote mucosal response [
11].