Mucosal immunity and vaccination against HIV (2002)

Background to mucosal immunity The critical feature of mucosal immunity to environmental agents in the gastro-intestinal and respiratory tracts is whether these antigens elicit an immune response or tolerance. The complex subject of tolerance is beyond the remit of this review and can be found elsewhere [1,2], but there is so far no evidence that tolerance can be induced via the genital or rectal mucosa. On the other hand, immunity has been elicited by direct immunization of the cervico-vaginal, rectal, and male urethral tracts reviewed in [3]. Unlike the well-defined inductive site, represented by Peyer's patches in the G-1 tract or lymphoid aggregation in the bronchial tract, corresponding structures have not been identified in the genital tract. However, tracing the migration of T and B cells with the PKH-26 dye has revealed that the internal and external iliac lymph nodes may function as an inductive site from which these cell migrate to the effector site, in the lamina propria of the cervico-vaginal mucosa and rectum [4]. The involvement of genital immunity within the mucosal associated lymphoid tissue (MALT) was documented by both oral and nasal immunization inducing antibodies in the genital tract [5]. A significant finding was that genital or rectal immunization elicits not only local mucosal but also systemic T and B cell immune responses [6–9]. Thus, mucosal or paramucosal (via the regional lymph nodes) immunization induces (i) mucosal immunity which is the first line of defence against HIV, (ii) regional lymph node immune responses which is the major inductive site and (iii) systemic immunity which reaches other parts of the body by the circulating blood (Fig. 1).Fig.1.: Mucosal or paramucosal (via the regional lymph nodes) immunization induces, mucosal immunity which is the first line of defence against HIV, regional lymph node immune responses which is the major inductive site, and systemic immunity which reaches other parts of the body by the circulating blood.Cells and receptors involved in mucosal immunity The first barrier in sexual transmission of HIV is the cervico-vaginal, rectal and oral epithelium. The mean thickness of the stratified squamous epithelium of the vaginal or oral mucosa is about 10 times greater than that of the columnar rectal epithelium. This is consistent with the rectal mucosa being more vulnerable to be breached during rectal intercourse than vaginal epithelium during vaginal sex. The stratified squamous epithelium of the foreskin and the pseudostratified squamous epithelium of the urethra may also become infected on exposure to HIV-infected secretions during heterosexual intercourse or on direct contact with infected rectal tissues during anal sex. HIV transmission through these epitheia and their constituent cells has not been studied extensively and will be briefly discussed below. Direct transmission or infection of cervical or intestinal cells has been demonstrated in vitro between HIV infected monocytes and epitheial cells [10]. Entry of HIV can take place in the absence of CD4+ cells and is mediated by the phagocytic pathway, through membrane invagination enclosing virions, or through receptormediated endocytosis. There is evidence that HIV binding is mediated by galactosyl ceramide on vaginal or rectal epithelial surface [11,12] and that the virus is transcytosed to the underlying lymphoid cells [13,14]. Langerhans cells (LC) and dendritic cells (DC) express the CD4 receptor and CCR5 coreceptor for HIV [15,16] and they are present in human cervico-vaginal, foreskin and oral but not rectal or urethral epithelia [17]. A large number of these cells were found in the foreskin and this may be one factor in determining the higher risk of uncircumsized males contracting HIV infection. Dendritic cells (DC) found beneath the rectal and vaginal epithelia may express DC-SIGN which binds HIV gp120 with high affinity and carries the virus to the regional lymph nodes [18,19]. Microfold (M) cells are found among the epithelial cells covering Peyer's patches in the intestine [20] and they have been reported in the rectal mucosa [21]. Rodent M cells are capable of transmitting HIV to lymphoid cells [22], but this has so far not been formally demonstrated in human M cells. Once HIV is transmitted through the cervico-vaginal or rectal mucosa, there are numerous CD4+ T cells, DC and macrophages in the lamina propria for HIV to infect. Genital infections, trauma and especially cervical erosions greatly enhance HIV infectivity. Mechanism of protection in vaginal and rectal HIV infection (a) Cells in Innate immunity Innate immunity has so far received only limited attention and has been recently reviewed [23]. DC and macrophages have been dealt with above; Type 1 IFN, TNFα and the complement system will not be considered here. NK cells function as effector cells in the early innate immune response and are regulated by a balance between inhibitory and triggering signals [24]. Inhibitory receptors specific for MHC class I molecules allow NK cells to respond to the loss of class I expression in viral infections. Humans employ Killer Immunoglobulin-like Receptors (KIR), the ILT2 LIR 1) molecule [25], and CD94/NKG2 lectin-like receptors specific for HLA-E. NK cells may exert a direct antiviral effect and promote an efficient Th1 response [26]. HIV selectively downregulates surface MHC class I expression (HLA-A and HLA-B) and antigen presentation to T lymphocytes [27]. yδ T cells may play a role in HIV infection, as they are involved in mucosal protection and may lyse HIV or SIV infected target cells [28]. yδ+ T cells also generate anti-viral suppressor factors, RANTES, MIP-1α and MIP-1β which can prevent SIV infection by binding to and downmodulating the CCR5 coreceptors [29]. In HIV infected humans, the Vδ1 subset of yδ+ T cells is upregulated [30,31]. We have investigated the protective potential of yδ+ T cells in macaques, challenged rectally by SIVmac 251 J5 molecular clone [29]. This revealed a significant increase in yδ T cells eluted from the rectal mucosa and the related iliac lymph nodes in protected as compared with infected macaques. (b) Soluble mediators involved in innate immunity to HIV infection Activation of CD8+ T cells by antigens or mitogens generates cell free HIV and SIV suppressor factors some of which have not been yet identified [32,33]. CC chemokines RANTES, MIP-1α and MIP-1β are produced by activation of macrophages, DC, T, NK and yδ T cells [34–36]. There is a great deal of evidence that the 3 CC chemokines can block the CCR5 coreceptors and prevent HIV infection in vitro [37] or SIV infection in vivo [38]. In vivo, the 3 chemokines were generated specifically by immunization with SIV gp120 and p27 in Alum [38,39], xenoimmunization in macaques [40] and alloimmunization in women [41]. There is evidence both in vitro [42] and in vivo [43] that raised concentrations of CC-chemokines downmodulate the cell-surface expression of CCR5. Resting or activated CD8-enriched cells eluted from normal rectal mucosa generate a large number of RANTES- and MIP-1β secreting cells [43]. This might be due to stimulation by the large number of gram negative and -positive bacteria residing in the rectum, as these contain lipopolysaccharides (LPS) and heat shock protein (HSP), both of which induce CC chemokines. Consistent with these findings is the increased concentration of RANTES, MIP-1α and MIP-1β secreted by the iliac compared with the axillary lymph node cells [39], as the former drain the rectal mucosa, with its indigenous microbial population, unlike the axillary lymph nodes which drain the relatively aseptic skin. Furthermore, the highest mRNA levels of CC chemokine were reported from lymph nodes draining the mucosal tissues [44]. Chemokines have been demonstrated in cervico-vaginal washings from HIV infected women [45]. The concentration and frequency of these chemokines were found in the following descending order: IL-8, RANTES and MIP-1α. The concentration of RANTES reached levels that inhibit HIV replication and might be protective at the surface of the cervico-vaginal mucosa [46]. SDF-1 (stromal derived factor) is the ligand of CXCR 4 which is expressed on naive T cells (CD4 or CD8 CD45RA+) and monocytes [47]. In vitro, SDF-1 binds and downmodulames CXCR4 [48]. High concentrations of SDF-1 are expressed constitutively in human cervico-vaginal and rectal epithelial cells and CXCR4 mRNA was readily detectable in these epithelial cells [49]. Quantitative RT-PCR studies showed that CCR5 is expressed 10 times more frequently than CXCR4 in cervical biopsies of women with and without sexually transmitted diseases [46]. However, greater proportion of both CCR5+ and CXCR4+ CD4 lymphocytes was found in normal human colonic mucosa [50]. (c) TH1-TH2 polarisation and the role of IL-12 TH1 and TH2 cytokines are generated by CD4 and CD8 T cells and TH1 cytokines are involved in protection against the development of AIDS [51]. TH1 cytokines, especially IL-12, are required for the cellular functions, especially of CD8+ cytotoxic cells and CD8+ suppressor factor against HIV [52]. Leucocyte traffic is regulated by IL-12 which is critical in inducing selectins and the α6β1 integrin to direct these cells to mucosal tissues [53]. It is significant therefore that IL-12 production may be inhibited in HIV infection [54,55]. The primary function of chemokine receptors is in trafficking of leucocytes and TH1 cells express CCR5 and CXCR3, whereas TH2 cells express CCR3, CCR4 and CCR8 [56–58]. Indeed, DNA encoding RANTES and MCP-1 induces TH1, whereas MIP-1α favours TH2 polarization [59]. Furthermore, MIP-1α, MIP-1β and RANTES induce DC to produce IFNγ and exert potent TH1 polarizing effect [60]. These results suggest that chemokine-receptor mediated activation might affect TH1-TH2 polarization. (d) Adaptive immunity Secretory IgA (SIgA) antibodies are an essential feature of mucosal immunity and are present both in vaginal and rectal secretions. However, IgG antibodies are also present in these, especially vaginal secretions. IgA antibody secreting cells in the iliac lymph nodes were significantly associated with protection against rectal mucosal SIV infection in macaques [38]. SIgA antibodies to HIV or gp160 have been found in vaginal secretions of sero-negative women highly exposed to HIV infection [61,62]. Some of these women have serum IgA and vaginal SIgA neutralizing antibodies [63,64]. Another mechanism that may be involved is SIgA or dimeric serum IgA antibodies to HIV, inhibiting transcytosis of HIV across human epithelial cells [13,64]. Serum IgG antibodies to CCR5 were identified in xenoimmunized macaques [65,66] and a comprehensive study showed that human CCR5 is immunogenic in macaques despite 98% identity with simian CCR5 [66]. IgA and IgG antibodies to the N terminal, first and second extracellular loops of CCR5 were elicited and can block CCR5-mediated HIV transmission. Antibodies to CCR5 can be induced by allo-immunization of women and are found in some multiparous women [41]. Antibodies to CCR5 were also reported in subjects with homozygous Δ32 CCR5 mutation [67] in sero-negative women at risk from HIV infection [68] and recently in normal serum IgG used in immunotherapy [69]. Furthermore, blocking of CCR5-mediated HIV transmission by CC chemokines can be enhanced by the complementary effect of antibodies to CCR5 [66]. Thus, a dual mechanism of blocking CCR5 could operate, in which the three CC chemokines and antibodies to CCR5 bind extracellular domains of CCR5, block the receptor function and prevent HIV or SIV transmission. However, immunization of macaques with DNA of CCR5 peptides alone failed to protect the animals from IV challenge with SIVmac [70]. Macaques immunized with baculovirus-generated CCR5 or those immunized with the CCR5 N terminal or loop 2 also failed to be protected from IV challenge with SIVmac 8980 (W Bogers et al., unpublished). Immunization with HIVgp120, SIVp27 and CCR5 peptides linked to HSP70 however, can elicit protection from SHIV 89.6P infection, and a decrease in viral load or clearance of the virus in macaques. T cell responses, especially CTL in systemic immunity has been well recognized but there is also convincing evidence that CTL in the cervico-vaginal mucosa may be involved in local protection against SIV in macaques [71,72]. CD4+ T cell helper function has been demonstrated in the iliac lymph nodes [38]. Cervical CD8+ CTL to HIV was found in discordant partners [61] and in HIV resistant sero-negative sex workers, as compared with low-risk control groups [62]. Although CTL were also found in blood, there was relative enrichment of the cervical CTL response. Thus, a complete array of SIgA antibodies, CD8+ CTL, CD4+ T cell help, cytokines and chemokines may be involved in the effector arm of immunity of the cervico-vaginal and rectal mucosa. Mucosal adjuvants Most vaccines, with the exception of replicating viruses require an adjuvant to initiate adaptive immunity. The mechanism of adjuvanticity is complex and outside the present remit, but has been reviewed by others [73,74]. The best defined mucosal adjuvant is cholera toxin which binds GM-1 receptors expressed on epithelial cells [75]. E. coli heat labile toxin (LT) is also a potent mucosal adjuvant [76] and binds GM-1. However, whilst CT induces predominantly TH2 responses LT may induce both TH1 and TH2 responses [77]. Microparticles of biodegradable polymers of polylactide-co-glycolides [78] CpG-DNA [79] and incorporation of cytokines or chemokines [80] are also important mucosal adjuvants. We have recently demonstrated mucosal adjuvanticity by HSP70 and this is dependent on stimulating production of CC chemokines [35]. These attract monocytes, immature DC, T cells and B cells which are the principal cells involved in generating an immune response [59,81]. The innate or CC chemokine generating component of HSP70 is linked with a specific peptide that resides in the pocket of HSP70 and elicits specific immunity HSP70 binds CD40 in stimulating macrophages and DC to generate CC chemokines [82] and IL-12 when the HSP70 is administered in the truncated form [83]. This function, linked with that of HSP70 translocating molecules from outside the cell into the HLA class I pathway enables HSP70 to elicit CTL, as well as IgG2a antibody responses. Mucosal routes of immunization Within the mucosal associated lymphoid tissue (MALT) any site might be used for mucosal immunization (Table 1). This concept was evolved in rodent experiments and has not been validated to the same extent in primates. Whilst oral or nasal immunization is the most convenient means of vaccine delivery, it may not elicit effective protecon against HIV. For effective immunity it is essential for the antigen to be localized to reach the draining lymph nodes in the optimum dose, as was demonstrated in a comprehensive investigation of mucosal immunization in non-human primates [3,6–9]. Rectal or cervico-vaginal immunization with SIV p27 antigen linked to cholera toxin B subunit (CTB) needed to be boosted by gastric immununization to induce local mucosal and systemic immunity in non-human primates. However, using CT alone elicited local mucosal and systemic IgA and IgG antibody secreting cells to CT [9]. Paramucosal (targeted iliac lymph node) immunization of macaques elicits potent secretory and systemic immune responses with subunit SIVgp 120 and p27 in Alum [38,39,84]. The rectal and vaginal mucosal routes of immunization have also been studied in humans and showed local and systemic IgA and IgG antibodies to CTB, though oral immunization also induced rectal and vaginal antibodies but at a lower titre [85]. Mucosal HIV-1 DNA administered by a jet injection showed that higher IgA antibodies in intestinal and lung washings were elicited by oral than nasal immunization [86]. This strategy was also carried out in HIV infected subjects by jet injection into the retro-molar mucosa of HIV nef, rev and tat (x3) [87]. The results suggested an increase in local CD4+ and CD8+ T cells, some of which expressed IL-2. It cannot, however be overemphasized that the results of immunization by different routes varies significantly from one species to another.Table 1: Routes of immunization eliciting mucosal immunity which can be boosted by IM immunization. The rectal or vaginal immunization can be enhanced by oral or nasal immunizationHIV is primarily a mucosal infection There is convincing evidence that the gut is the primary target of HIV infection, even when infection is by the IV route [88]. Within days of IV infection SIV infects CD4+ memory T cells in the intestine and this coincides with the loss of these cells, before corresponding changes are found in the peripheral lymphoid tissues. An important conclusion was drawn that the acute phase of SIV (or HIV) infection is primarily a disease of the mucosal immune system and that preventive vaccination strategies should target the mucosal immune system. The SIV specific CD8+ T cells which express the mucosal homing receptor α4β7 were detected in the intestine and PBMC of macaques infected with attenuated SIV [89]. Mucosal selection of predominantly CCR5 utilizing strains of HIV has not been fully accounted for. Cervico-vaginal and foreskin epithelia have a large number of Langerhans cells [17] which may express CD4 and CCR5 cell-surface receptors [90]. CCR5 is not expressed by cervical epithelial cells, but mononuclear cells show 10-fold greater number of CCR5 than CXCR4 expressing cells and CCR5 was significantly increased in women with sexually transmitted diseases [91], thereby promoting selective transmission of R5-HIV. High concentrations of SDF-1 found in genital epithelial cells may block and downmodulate CXCR4, thus preventing X4-dependent HIV transmission [49]. Epithelial transcytosis is mediated by galactosyl cerebroside that is expressed on the surface of endometrial (HEC-1) or intestinal (HT-29) epithelial cell lines and does not differentiate between the R5 and X4 strains of HIV However, primary epithelial cells from the intestine express and CCR5 which selectively R5 but not X4 of HIV to CD4+ T cells in the lamina propria this selective mechanism also to rectal mucosa has not been However, cells in the genital or rectal mucosa not to differentiate between R5 and X4 dependent HIV in their binding and of HIV-1 to the regional lymph nodes they the virus to CD4+ T cells The relative of cell surface expression of CCR5, CXCR4, galactosyl cerebroside and chemokines in HIV transmission of mucosal tissues is not mucosal immunization of HIV have recently been and the present will be to mucosal immunization. experiments in macaques have in the a that HIV is a mucosal infection. this in studies macaques were immunized by the systemic route but challenged with SIV or SHIV by the vaginal or rectal mucosal This to the 3 major strategies of preventive immunization. (i) DNA (ii) expression of HIV in viral and (iii) DNA viral Mucosal challenge a significant in the by the to the mucosal system which a and However, it to the principal that of HIV infection by the vaginal or rectal route may require mucosal in to the inductive and effector of the mucosal immune system. This was in the mucosal lymph nodes of macaques with and p27 in Alum and rectal mucosal challenge with SIVmac [38]. of SIV infection was induced in macaques, with 4 out of complete protection and the 3 macaques a decrease in viral load or as compared with of being This strategy was recently in in which infection by the rectal route was by vaccination with cells studies the of the lymph nodes as an effective site of immunization is being investigated at et al., unpublished). This HIV and to whether immunization greater mucosal immune responses than immunization into the A of the of mucosal immunization was rectal of 4 peptides of HIV helper and SIV or CTL with E. toxin (LT) Rectal challenge with in lower viral or clearance of the virus in the immunized as compared with the control macaques. A of immunized macaques failed to the and showed significant levels of the virus in the in to the rectally immunized animals which showed no detectable virus in the or paramucosal immunization with HIV SIV p27 and CCR5 linked to the HSP70 adjuvant also elicited significant clearance of SHIV in 4 of macaques, compared with of 4 et al., SHIV 89.6P has to be a CXCR4 binding and the vaccine was predominantly CCR5 it is that this immunization strategy may to be more effective against R5 strains of Mucosal immunization but IV challenge was in in macaques with oral (or immunization with a expressing SIV and and challenged IV with SHIV 89.6P infection of immunized and control the immunized macaques showed or whereas the showed high virus levels and of macaques Although viral load and of HIV is a of in the of infection be the in of the evidence of of infection, even when the virus is and attenuated as or have been studied as for DNA administered by the oral It is also that of high of antibodies to and vaginal or oral mucosal infection with SHIV and Mucosal immunity at the site of HIV effector SIgA and IgG antibodies, with CD8+ CTL, T cells, cytokines and chemokines. The inductive mechanism of immunity elicited at the mucosal site Langerhans cells in the cervico-vaginal mucosa and DC in the of the and rectal mucosa. The antigen is carried to the regional lymph nodes by the and DC to present the antigen to CD4+ T cells which generating B cell antibodies, suppressor factors and chemokines. The protective mechanism may both antibody and cellular effector responses to HIV CCR5 coreceptors and galactosyl cerebroside on epithelial cells. Thus, HIV can be mucosal tissues by immune responses to HIV and those to the as well as innate cells and In significant systemic are elicited in the circulating that HIV or SIV is a predominantly mucosal infection, it is important to a mucosal strategy of immunization. The to the from and which to out some of the studies in the