1. Field of the Invention
The invention in the field of immunology and infectious disease relates to novel peptide immunogens from a random library selected by an antibody against a Chlamydial glycolipid exoantigen (GLXA) or corresponding to antigen-binding regions of an anti-idiotypic antibody (mAb2) specific for an anti-GLXA antibody (Ab1) and which serves as a molecular mimic of GLXA, and their use in inducing antibodies against GLXA—a genus-wide (“genus-specific”) chlamydial antigen.
2. Description of the Background Art
More than 1 million new cases of chlamydial infection were reported in 2006, and cost the economy over $1 billion dollars. Despite increased surveillance and treatment, chlamydial sexually transmitted disease (STD) infections continue to rise. Chlamydia trachomatis is the leading cause of tubal infertility and pelvic inflammatory disease (1,2). Asymptomatic and undiagnosed chlamydial infections are estimated to double the reported rate of infections. Chlamydial genital tract infection is more than 5 times more common than gonorrhea (3) and has been correlated with increased risk of infection with HIV and other STD pathogens (4). Chlamydial genital infection occurs in 5-15% of pregnant women, and 50% of their babies will develop inclusion conjunctivitis or respiratory infections (5) making C. trachomatis the most common ocular pathogen in infants (6). In sexually transmitted chlamydial infections, other factors such as repeated exposure, asymptomatic (unapparent) and/or persistent infections make diagnosis difficult. Although antibiotics can clear many chlamydial infections, they do not prevent re-infection.
In vitro antibiotics can drive Chlamydiae into a persistent, nonculturable state (7). Persistently infected cells in vitro are resistant to azithromycin (8). Animal studies suggest (9) that early antibiotic treatment may interfere with the development of some natural protective immunity, and thus pre-dispose patients to more extensive pathology associated with pelvic inflammatory disease and worse sequelae. Genital infections also predispose to development of a significant proportion of reactive arthritis cases in which viable, metabolically active organism is present in synovium (10, 11). For recent reviews on Chlamydiae, see, for example, Ref 12).
Trachoma, the leading cause of infectious blindness in humans (13, 14), is caused by repeated ocular infection with ocular biovars of C. trachomatis. Of the tens of millions of people suffer from trachoma, up to one-fourth become blind. Trachoma has largely disappeared from North America and Europe, where extraocular chlamydial infections remain of great importance. Chlamydia pneumoniae (Cpn), a cause of community-acquired pneumonia in adults, has been associated with atherosclerosis (15,16); seroepidemiologic studies suggest that the majority of adults have been exposed to Cpn. Cpn has been associated with other chronic inflammatory diseases including late onset Alzheimer's disease (17, 18), one or more forms of multiple sclerosis (19, 20), and temperomandibular joint disease (TMJD) (21, 22, 23) A link between atherosclerosis and Alzheimer's disease (AD) is suspected in some cases (e.g., 24).
C. psittaci infects avian species and can have major economic impact on the poultry industry, affecting not only production, but also endangering poultry handlers (25). Thus, the public health significance of chlamydial infection is enormous. A genus-specific protective vaccine with broad protective capacity beyond selected serovars of C trachomatis would have great value.
Nanoencapsulation and Delivery of Vaccine Candidates.
Novel delivery methods for vaccine candidates have been developed over the past decade. With the advent of nanotechnology and “nanomedicine,” therapeutic uses for nanoparticles (NP) has rapidly expanded. The present inventors and colleagues reported on their use of poly(lactic-co-glycolic acid (PLGA) microsphere-encapsulated protective antibodies as a chlamydial vaccine which was delivered orally and intranasally (26, 27). The present inventors and colleagues have recently found that nanoparticles are rapidly taken up into Chlamydia-infected cells in vitro, and that nanoparticles can be targeted to infected tissues (e.g., 28,29,30). Others have shown that PLGA nanoparticles can be used to deliver peptides, oligomers (DNA) or drugs in vivo (31-36). NP formulations with alternative polymers such as chitosan or alginate have been successful for mucosal delivery (31,37). The effects of the size and surface characteristics of the NPs have been investigated, (38, 39)
The present inventors and colleagues originally tested their first vaccine candidate in microspheres in part because nanosized materials for similar drug and peptide delivery were not yet available. Encapsulation has at least two major advantages: (1) an encapsulated vaccine antigen (“Ag”) such as a monoclonal antibody (mAb) or a peptide or polynucleotide could be delivered orally without loss of function because of protection from gastric acids. Alternatively, intranasally or trans-tracheally delivered antigens in NPs would remain in the nasopharynx or lungs long enough to enter local antigen-presenting cells such as lung macrophage or dendritic cells (DC).
The 1990's dogma was that uptake of particle-based vaccines/antigens to mucosally immunize via uptake at Peyer's patches required particles with diameters of 1-10 μm (40, 138). Since then, Amidi et al., (31), Saltzman and others have demonstrated that NPs (<500 nm diameter) could not only successfully be delivered mucosally and immunize against the Ag delivered, but could be more efficient. Part of the latter success is due to Ag-loaded NPs inducing DC maturation (36); NPs are efficiently taken up both by DCs and macrophages (141). NP size delivery vehicles remain under study (38, 41, 137) and the potential for newer materials and NP designs have broadened their appeal as vaccine delivery vehicles.
PLGA co-polymer is FDA-approved for human use (dissolving sutures) and acts as a slow delivery device compared to free Ag, besides its adjuvant properties (42). PLGA NPs can be (a) fluorescently labeled to follow uptake in cells and tissues, (b) targeted to specific types of cells, and (c) conjugated to polyethylene glycol (PEG), also known as “pegylation” to sustain their circulating half life. Presumably nanosized particles containing vaccine candidates can be taken up at sites other than the Peyer's patches, probably by pinocytosis into enterocytes or DCs which locally sample the gut or other mucosal surface for foreign Ags. Upon recognition and uptake by DC, these Ag-presenting cells travel to the regional draining lymph nodes; Ag released from NPs inside the DC will be presented to T lymphocytes. This activates T cells which respond upon subsequent exposure to the immunizing Ag (or the whole organism, in this case, C. trachomatis). Such responses are required to clear infectious organisms from the mucosal sites.
Chlamydial Biology and Vaccine Targets
Chlamydiae are complex, obligate intracellular bacteria with a biphasic developmental cycle: (a) the elementary body (EB) which is infectious but metabolically inactive like a spore and (b) the reticulate body (RB) which is non-infectious but metabolically active. A schematic representation of the developmental cycle is shown in FIG. 1. A simple view is that immune responses to both the extracellular EB via antibody (“Ab”) and intracellular stages (RB and EB), plus responses to the persistent form of “aberrant bodies” (“AB”) via potent CD4 T cell responses and perhaps CD8 cytotoxic T cells are required for the “perfect” vaccine.
FIG. 2 is a schematic drawing depicting the earlier mAb2 vaccine candidate which was delivered in microparticles (26,27) and its replacement by peptide mimetics.
Novel vaccine strategies are needed for chlamydial infections as traditional approaches with purified Ag or recombinant peptides have failed to protect, despite their immunogenicity (46, 47). Some of the difficulty in designing a protective vaccine approach relates to the use of a variety of different animal models. Newer molecular and biochemical methodologies have provided highly immunogenic Ag constructs/peptides which may induce protective cytotoxic T lymphocyte (CTL) responses (48), allow novel DNA vaccine constructs for the “major outer membrane protein” (MOMP) Ag or tests of new adjuvants such as CpG, (47, 49, 50)). An alternative approach adopted by the present inventors, is to use peptides derived by standard, accepted methods as vaccine candidates. During the past 10 years, peptides with sequences derived from anti-idiotypic (Anti-Id) Abs (which include mAbs) or conventional mAbs were shown to immunize or protect against several infectious agents and have been used extensively for cancer vaccine development (142-144).
Anti-Chlamydial Immunity can be Protective or Pathogenic
Primary chlamydial infection does not lead to lasting immunity against subsequent re-infection (51-53). The immunopathogenic responses to infection complicate vaccine development. After primary infection, part of the local immune response to re-infection appears to be a destructive local CD4+ T cell-mediated delayed-type hypersensitivity (DTH) response to hsp60 or to another chlamydial Ag (54-58).
The complex immunology of chlamydial infection has been extensively studied in several models (60), but the cellular and molecular requirements for protective immunity remain largely unelucidated. DCs pulsed with MOMP peptides appeared immunogenic, but failed to protect against C. muridarum (MoPn) genital challenge even though DC delivery of killed MoPn was protective (59, 60). Igietseme et al. (61) showed protection in mice immunized with EB-pulsed DC obtained from IL-10 knockout (KO) donors, and that DC with the IL10KO more rapidly stimulated Th1 responses in an IFNγ-dependent manner. This group showed earlier that chlamydial Ag-Ab complexes increased DC uptake of Ag via engagement of the cells' FcR to generate better effector responses in vitro and in vivo (62, 103). These results complement other studies showing that Ags directed to APCs via FcR engagement can shift pro-inflammatory immune responses to anti-inflammatory immune responses to those same Ags (63,64). Coupled with recent results of Morrison (79) regarding an important B cell component to CD4-mediated clearance of infection, it is now clear that both T and B cells are required for anti-chlamydial protective immunity.
Mucosal immune responses to Chlamydia, including neutralizing Ab, are believed to be required for protection from infection although presence of neutralizing Ab alone does not assure protective immunization, presumably in part because of the chlamydial Ag targeted. Vigorous Ab responses to numerous chlamydial Ags, such as MOMP, a Chlamydia -secreted protease factor designated CPAF and lipopolysaccharide (LPS), measured in sera or secretions of infected individuals supported the vaccine potential of one or more of the latter, and most of these have been tested with varying success, e.g., (47, 49, 65. 66). An LPS-based vaccine was not protective although LPS is genus-specific (145). MOMP based vaccines are serovar-specific, in contrast to the genus-wide protective immunogens of the present invention, and would require cocktail vaccine approaches.
The genus-specific, secreted chlamydial glycolipid exoantigen (“GLXA”), which is distinct from LPS (67-74), is an immunogenic and also an immunologically relevant a target. Abs from patients infected with C. trachomatis, C. psittaci, and Cpn react to GLXA (81). Many anti-chlamydial immune responses are T cell-dependent. Specific T cell responses to MOMP and other Ag have been shown, and CD4 cells have a role in clearance (75-80).
Recent new chlamydial Ags include those identified by proteomic screening of patient samples (81). Barker et al. (82) recently showed a chlamydial T cell antigen, NrdB representing a ribonucleotide reductase small chain protein. Karunakaran et al. (83) used immunoproteomics to identify novel peptides bound by MHC Class I or II molecules with the C. muridarum mouse model. Cytokine/chemokine responses to the MoPn and other serovars suggest that activation of both Th1 and Th2 CD4 cells are important in clearance (84-87)). However, higher levels of IL-10 have been related to susceptibility to MoPn (88). Shifts in dominant Th have been associated with protection against other intracellular pathogens such as Leishmania and Mycobacteria (89-91), but this effect has yet to be been shown for any chlamydial vaccine candidate. The mAb2-induced isotype shifts in anti-GLXA Ab3 suggest the anti-Id vaccine induces both Th1 and Th2 cell-mediated anti-GLXA responses which are profoundly affected by the route of immunization.
According to the present invention, the protective peptide vaccine candidates with the appropriate Th and CTL epitopes will induce both Th1 and Th2 responses and probably CD8+ CTL responses, respectively.
Most of the expected responder/effector cells and their cytokines have been found during chlamydial infection and clearance (85, 92). However, these immunohistochemical (IHC) approaches have been focused on innate and adaptive immune responses to infection rather than on responses to vaccination. Studies with transgenic (Tg) and KO mice have suggested that MHC Class II+ T cells are critical in chlamydial (MoPn) clearance, whereas T cells involved in MHC Class I pathway are not (93). It is more likely that a continuum of Th1 vs Th2-associated responses occurs (94, 95)), and many factors including Ag-processing pathway(s) (96) influence the outcome.
A potential protective mechanism in chronic chlamydial inflammatory disease is mediated by regulation of pro-inflammatory Th1 cell and monocyte/macrophage/DC responses. Roles for CD8+ T cells in responses to this intracellular pathogen have long been suggested, and evidence for CD8+ CTL against both C. trachomatis and Cpn has been published (48,97-99). However, immunogenic and protective peptides that induce CD8 responses across serovars or species have not yet been demonstrated. Manipulation of APC, particularly DCs pulsed with (UV)-EB induced varying levels of protective immunity. For example, DC exposed to live EB acquired a more mature DC phenotype than that seen with UV-EB and produced higher levels of IL-12 which would enhance CD4 Th1 responses (113, 114).
Development of chlamydial vaccines development requires                (1) identification of one or more target Ags,        (2) induction of better protective responses to overcome pathogenic immune responses, and        (3) lasting protection against primary, secondary, and heterologous infections in one or more animal models.        
Real clinical exposures to Chlamydia are presumably low dose and thus minimally immunogenic (until in vivo replication begins). So care is required in interpreting evidence of immune responses to large challenge doses in animal models as these may reflect multiple pathways of stimulation which differ from more subtle responses to natural infection. Since previous infection alone does not induce fully protective immunity in humans, and because single infections are usually self-limited, it is even more important to identify and induce immune responses which go beyond those described above without exacerbating the inflammatory component. A new question has been articulated recently in response to the observation that early antibiotic treatment of chlamydial infections may abrogate development of some natural protective immunity, and in this way could lead to worse late sequelae such as infertility (146, 147).
On the other hand, natural clearance of organism may not represent the required response(s) for protective immunity. Do highly immunodominant Ags obscure potentially protective responses to other Ags? Achieving a balance between protective and pathogenic immunization is important for a vaccine for human populations that are continuously re-exposed or were previously exposed to Chlamydia. Understanding how to inhibit dissemination and establishment of chronic infections at nonmucosal sites, and the effect of any anti-chlamydial vaccination on these events are critically important. The present invention identifies the effect of peptide immunogens, such as those derived from the sequence of mAb2 variable regions on such a balance and on disseminated chlamydial infection which reflects human disease.
Chlamydia trachomatis and Animal Models of Disseminated Infection
A new appreciation has emerged recently about the dissemination phase of chlamydial infections. Circulating cells (probably monocytes and/or monocyte-derived DCs) traffic and collect, or are trapped, at one or more sites. A common site for C. trachomatis dissemination is the synovium, and indeed, a subset of patients develops reactive arthritis (ReA). Chlamydiae are the only viable and metabolically active bacteria in ReA synovium, and are in a molecularly-defined persistent form (as to morphology and gene expression) when patients present to the rheumatologist (10, 100-107).
The synovium has been postulated to be a site of entrapment of infectious organisms, circulating particulates, etc. IHC and immunoelectron microscopic studies showed that both intact Chlamydia and chlamydial Ags are present in the ReA synovium, ((110, 11)). However, isolation of culturable Chlamydia from joints was reported only once (112); most attempts failed (106)). Under some conditions, C. trachomatis generates persistent infection (10, 101, 107, 113-116), though very low levels of EB are produced, and a number of genes encoding MOMP, chsp60, ftsK, ftsW, etc. are either down- or up-regulated.
Many groups, including the present inventors have developed PCR-based Chlamydia detection systems, (117-122). With the publication of genomes for several C trachomatis serovars, PCR/qPCR for additional chlamydial gene transcripts has become possible. The C. trachomatis genome project has enabled the present inventors' own studies of selected chlamydial genes expected to be aberrantly expressed when the organism enters a persistent state. Targeting selected genes involved in specific stages of chlamydial development and differentiation indicates that chlamydial gene expression in actively infected cells differs significantly from that observed in ReA synovial tissues and in persistently infected human monocytes in vitro (118,123). Remarkably few animal studies have investigated Chlamydia-associated ReA.
The present inventors and colleagues were the first to show vaccine-mediated reduction in experimental ReA in mice. Initially, ocular infection of mouse conjunctivae (an ocular mucosal tissue) resulted in chlamydial dissemination to synovium (124). More recently, the present inventors focused on a genital infection model—more representative of human Chlamydia-associated ReA cases in the US and Europe. In the latter models C. trachomatis dissemination to synovial tissues and consequent knee pathology were documented.
An overview of the synovial inflammation induced in the present inventors' murine ocular and genital infection models has been published (124-126). Chlamydial dissemination occurs in other animal models: Cpn was shown (127), to disseminate to distant sites after intranasal challenge of mice or after transfer of infected PEC, but neither synovium nor the CNS was assayed. Studies (128) with MoPn-induced genital infection resulted in an acute arthritis. The latter studies utilized either presensitization or intra-articular chlamydial challenge, making them a poorer mimic of natural dissemination from a genital infection. The same group (129) showed dissemination of GPIC (Chlamydiophila pecorum) from genital tract to joint in guinea pigs. A recent inbred rat model of chlamydial ReA (130) utilizes intra-articular injection of synoviocytes infected with C. trachomatis. While allowing examination of some questions relevant to ReA, it differs fundamentally from natural human infections in which the initial infected cell is not fibroblastic, nor would this be the host cell involved in chlamydial dissemination to joints. Therefore, the present inventors' model for C. trachomatis-associated ReA is advantageous for developing and testing of the vaccines of the present invention, and most particularly for study-mediated reduction of chlamydial ReA and synovial infection because of its noninvasive mode of disease generation.
The present inventors' Identification of an effective vaccine coupled with an effective delivery strategy to protect against chlamydial infections should have enormous public health impact worldwide. The encapsulation of immunogenic peptides into biodegradable NPs will facilitate better mucosal vaccination, help reduce cold chain requirements This invention represents novel approaches to prevention of Chlamydia-associated diseases, as nanotechnology has not been applied previously to studies of Chlamydia. Further, the approaches developed in accordance with this invention will serve as a basis for the development of vaccine formulations for other intracellular human pathogens.
There currently is no protective chlamydial vaccine. Sexually transmitted infections are largely asymptomatic in women and this can lead to ascending infections, pelvic inflammatory disease, ectopic pregnancies and infertility. Despite widespread screening and treatment programs, the numbers of cases of chlamydial sexually transmitted infections (STI) are still increasing and represent over one million new STI cases/year in 2007. Because these antigenic epitopes are genus-specific (genus-wide), not serovar-specific or supposedly biovar-specific (C. trachomatis vs. C. pneumoniae vs C. psittaci) the present vaccine compositions should protect against STI, cardiovascular disease, chlamydial pneumonia, some subsets of Alzheimer's disease and multiple sclerosis, not to mention chronic inflammatory disease sequelae like infertility.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.
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