Large intestine-targeted, nanoparticle-releasing oral vaccine to control genitorectal viral infection.

Both rectal and vaginal mucosal surfaces serve as transmission routes for pathogenic microorganisms. Vaccination through large intestinal mucosa, previously proven protective for both of these mucosal sites in animal studies, can be achieved successfully by direct intracolorectal (i.c.r.) administration, but this route is clinically impractical. Oral vaccine delivery seems preferable but runs the risk of the vaccine's destruction in the upper gastrointestinal tract. Therefore, we designed a large intestine-targeted oral delivery with pH-dependent microparticles containing vaccine nanoparticles, which induced colorectal immunity in mice comparably to colorectal vaccination and protected against rectal and vaginal viral challenge. Conversely, vaccine targeted to the small intestine induced only small intestinal immunity and provided no rectal or vaginal protection, demonstrating functional compartmentalization within the gut mucosal immune system. Therefore, using this oral vaccine delivery system to target the large intestine, but not the small intestine, may represent a feasible new strategy for immune protection of rectal and vaginal mucosa.

TLR agonists and/or IL-15 adjuvanted mucosal SIV vaccine reduced gut CD4⁺ memory T cell loss in SIVmac251-challenged rhesus macaques.

Adjuvant plays an important role in increasing and directing vaccine-induced immune responses. In a previous study, we found that a mucosal SIV vaccine using a combination of IL-15 and TLR agonists as adjuvant mediated partial protection against SIVmac251 rectal challenge, whereas neither IL-15 nor TLR agonists alone as an adjuvant impacted the plasma viral loads. In this study, dissociation of CD4(+) T cell preservation with viral loads was observed in the animals vaccinated with adjuvants. Significantly higher levels of memory CD4(+) T cell numbers were preserved after SIVmac251 infection in the colons of the animals vaccinated with vaccine containing any of these adjuvants compared to no adjuvant. When we measured the viral-specific CD8(+) tetramer responses in the colon lamina propria, we found significantly higher levels of gag, tat, and pol epitope tetramer(+) T cell responses in these animals compared to ones without adjuvant, even if some of the animals had similarly high viral loads. Furthermore, this CD4(+) T preservation was positively correlated with increased levels of gag and Tat, but not pol tetramer(+) T cell responses, and inversely correlated with beta-chemokine expression. The pre-challenged APOBEC3G expression level, which has previously been shown inversely associated with viral loads, was further found positively correlated with CD4(+) T cell number preservation. Overall, these data highlight one unrecognized role of adjuvant in HIV vaccine development, and show that vaccines can produce a surprising discordance between CD4(+) T cell levels and SIV viral load.

Innate and adaptive immune correlates of vaccine and adjuvant-induced control of mucosal transmission of SIV in macaques.

Adjuvant effects on innate as well as adaptive immunity may be critical for inducing protection against mucosal HIV and simian immunodeficiency virus (SIV) exposure. We therefore studied effects of Toll-like receptor agonists and IL-15 as mucosal adjuvants on both innate and adaptive immunity in a peptide/poxvirus HIV/SIV mucosal vaccine in macaques, and made three critical observations regarding both innate and adaptive correlates of protection: (i) adjuvant-alone without vaccine antigen impacted the intrarectal SIVmac251 challenge outcome, correlating with surprisingly long-lived APOBEC3G (A3G)-mediated innate immunity; in addition, even among animals receiving vaccine with adjuvants, viral load correlated inversely with A3G levels; (ii) a surprising threshold-like effect existed for vaccine-induced adaptive immunity control of viral load, and only antigen-specific polyfunctional CD8(+) T cells correlated with protection, not tetramer(+) T cells, demonstrating the importance of T-cell quality; (iii) synergy was observed between Toll-like receptor agonists and IL-15 for driving adaptive responses through the up-regulation of IL-15Ralpha, which can present IL-15 in trans, as well as for driving the innate A3G response. Thus, strategic use of molecular adjuvants can provide better mucosal protection through induction of both innate and adaptive immunity.

Using 3 TLR ligands as a combination adjuvant induces qualitative changes in T cell responses needed for antiviral protection in mice.

TLR ligands are promising candidates for the development of novel vaccine adjuvants that can elicit protective immunity against emerging infectious diseases. Adjuvants have been used most frequently to increase the quantity of an immune response. However, the quality of a T cell response can be more important than its quantity. Stimulating certain pairs of TLRs induces a synergistic response in terms of activating dendritic cells and eliciting/enhancing T cell responses through clonal expansion, which increases the number of responding T cells. Here, we have found that utilizing ligands for 3 TLRs (TLR2/6, TLR3, and TLR9) greatly increased the protective efficacy of vaccination with an HIV envelope peptide in mice when compared with using ligands for only any 2 of these TLRs; surprisingly, increased protection was induced without a marked increase in the number of peptide-specific T cells. Rather, the combination of these 3 TLR ligands augmented the quality of the T cell responses primarily by amplifying their functional avidity for the antigen, which was necessary for clearance of virus. The triple combination increased production of DC IL-15 along with its receptor, IL-15Ralpha, which contributed to high avidity, and decreased expression of programmed death-ligand 1 and induction of Tregs. Therefore, selective TLR ligand combinations can increase protective efficacy by increasing the quality rather than the quantity of T cell responses.

Reactivation of human herpesvirus-6 in natalizumab treated multiple sclerosis patients.

The alpha(4) integrin antagonist natalizumab was shown to be effective in patients with immune-mediated disorders but was unexpectedly associated with JC polyomavirus associated progressive multifocal leukoencephalopathy (PML) in two multiple sclerosis (MS) and one Crohn's disease patients. Impaired immune surveillance due to natalizumab treatment may have contributed to the JCV reactivation. As HHV-6 has been suggested to play a role in MS, we asked whether this virus could also have been reactivated during natalizumab therapy. Matched sera and CSF from a limited set of MS patients treated with and without natalizumab were examined for evidence of HHV-6. In addition, we also superinfected a persistent JC virus infected glial cell with HHV-6A to determine if JC virus can be increased. Elevated serum HHV6 IgG and HHV-6A DNA was detected in the CSF of a subset of patients but not controls. We confirmed that superinfection with HHV-6 of a JC virus infected glial cells increased expression of JCV. These results support the hypothesis that treatment with natalizumab may be associated with reduced immune surveillance resulting in reactivation of viruses associated with MS pathogenesis.

Estimation of low frequency antigen-presenting cells with a novel RELISPOT assay.

Adequate presentation of self and foreign antigens is a key factor for efficient T-cell immunosurveillance against pathogens and tumors. Cells presenting foreign antigens usually comprise a rare population and are difficult to detect even at the peak of infection. Here we demonstrate a CD8(+) T-cell-based approach that allows detection of specific antigen-presenting cells (APC) at a frequency of less than 0.0005%. When T cells are in excess, they form rosettes with rare APCs, which appear as single spots in an IFN-gamma ELISPOT assay. Using this RELISPOT (Rosette ELISPOT) method we demonstrate the dynamic interplay between CD8 T cells and professional and non-professional APCs following virus challenge.

T cell receptor recognition via cooperative conformational plasticity.

Although T cell receptor cross-reactivity is a fundamental property of the immune system and is implicated in numerous autoimmune pathologies, the molecular mechanisms by which T cell receptors can recognize and respond to diverse ligands are incompletely understood. In the current study we examined the response of the human T cell lymphotropic virus-1 (HTLV-1) Tax-specific T cell receptor (TCR) A6 to a panel of structurally distinct haptens coupled to the Tax 11-19 peptide with a lysine substitution at position 5 (Tax5K, LLFG[K-hapten]PVYV). The A6 TCR could cross-reactively recognize one of these haptenated peptides, Tax-5K-4-(3-Indolyl)-butyric acid (IBA), presented by HLA-A*0201. The crystal structures of Tax5K-IBA/HLA-A2 free and in complex with A6 reveal that binding is mediated by a mechanism of cooperative conformational plasticity involving conformational changes on both sides of the protein-protein interface, including the TCR complementarity determining region (CDR) loops, Valpha/Vbeta domain orientation, and the hapten-modified peptide. Our findings illustrate the complex role that protein dynamics can play in TCR cross-reactivity and highlight that T cell receptor recognition of ligand can be achieved through diverse and complex molecular mechanisms that can occur simultaneously in the interface, not limited to molecular mimicry and CDR loop shifts.

Extensive T cell receptor cross-reactivity on structurally diverse haptenated peptides presented by HLA-A2.

Previous studies have shown that individual TCRs are able to effectively recognize multiple peptide/MHC complexes that have varying degrees of structural diversity. These TCR cross-reactivities have usually been demonstrated by using peptides that have different amino acid sequences. To further examine the extent to which TCRs can accommodate structurally diverse ligands, we analyzed human TCR cross-reactivity to eight structurally distinct haptens that are coupled to the HLA-A2-binding Tax peptide with a lysine substitution at position 5 (Tax-5K, LLFG[K-hapten]PVYV). The results demonstrate that 71% percent of the haptenated-peptide-induced CTL lines could cross-react on at least one other hapten. We compared the effects of HLA-A2 mutants with substitutions at known TCR contact sites for recognition by hapten-cross-reactive CTL. Recognition of the A2 mutants was remarkably similar whether they were presenting the immunizing or the cross-reactive peptide, indicating that similar amino acid contacts are made by the TCR during recognition of both complexes. Thus, hapten cross-reactivity is apparently accomplished without major adjustments to the interaction between the TCR and the surface of the HLA-A2 molecule. Collectively, these results suggest that TCRs possess the molecular flexibility to accommodate very structurally diverse ligands while retaining conserved interactions with the surface of the MHC molecule.

Unraveling a hotspot for TCR recognition on HLA-A2: evidence against the existence of peptide-independent TCR binding determinants.

T cell receptor (TCR) recognition of peptide takes place in the context of the major histocompatibility complex (MHC) molecule, which accounts for approximately two-thirds of the peptide/MHC buried surface. Using the class I MHC HLA-A2 and a large panel of mutants, we have previously shown that surface mutations that disrupt TCR recognition vary with the identity of the peptide. The single exception is Lys66 on the HLA-A2 alpha1 helix, which when mutated to alanine disrupts recognition for 93% of over 250 different T cell clones or lines, independent of which peptide is bound. Thus, Lys66 could serve as a peptide-independent TCR binding determinant. Here, we have examined the role of Lys66 in TCR recognition of HLA-A2 in detail. The structure of a peptide/HLA-A2 molecule with the K66A mutation indicates that although the mutation induces no major structural changes, it results in the exposure of a negatively charged glutamate (Glu63) underneath Lys66. Concurrent replacement of Glu63 with glutamine restores TCR binding and function for T cells specific for five different peptides presented by HLA-A2. Thus, the positive charge on Lys66 does not serve to guide all TCRs onto the HLA-A2 molecule in a manner required for productive signaling. Furthermore, electrostatic calculations indicate that Lys66 does not contribute to the stability of two TCR-peptide/HLA-A2 complexes. Our findings are consistent with the notion that each TCR arrives at a unique solution of how to bind a peptide/MHC, most strongly influenced by the chemical and structural features of the bound peptide. This would not rule out an intrinsic affinity of TCRs for MHC molecules achieved through multiple weak interactions, but for HLA-A2 the collective mutational data place limits on the role of any single MHC amino acid side-chain in driving TCR binding in a peptide-independent fashion.

Strategic mutations in the class I major histocompatibility complex HLA-A2 independently affect both peptide binding and T cell receptor recognition.

Mutational studies of T cell receptor (TCR) contact residues on the surface of the human class I major histocompatibility complex (MHC) molecule HLA-A2 have identified a "functional hot spot" that comprises Arg(65) and Lys(66) and is involved in recognition by most peptide-specific HLA-A2-restricted TCRs. Although there is a significant amount of functional data on the effects of mutations at these positions, there is comparatively little biochemical information that could illuminate their mode of action. Here, we have used a combination of fluorescence anisotropy, functional assays, and Biacore binding experiments to examine the effects of mutations at these positions on the peptide-MHC interaction and TCR recognition. The results indicate that mutations at both position 65 and position 66 influence peptide binding by HLA-A2 to various extents. In particular, mutations at position 66 result in significantly increased peptide dissociation rates. However, these effects are independent of their effects on TCR recognition, and the Arg(65)-Lys(66) region thus represents a true "hot spot" for TCR recognition. We also made the observation that in vitro T cell reactivity does not scale with the half-life of the peptide-MHC complex, as is often assumed. Finally, position 66 is implicated in the "dual recognition" of both peptide and TCR, emphasizing the multiple roles of the class I MHC peptide-binding domain.

Tax and M1 peptide/HLA-A2-specific Fabs and T cell receptors recognize nonidentical structural features on peptide/HLA-A2 complexes.

Both TCRs and Ab molecules are capable of MHC-restricted recognition of peptide/MHC complexes. However, such MHC restriction is the predominant mode of recognition by T cells, but is extremely rare for B cells. The present study asks whether the dichotomy in Ag recognition modes of T and B cells could be due to fundamental differences in the methods by which TCRs and Abs recognize peptide/MHC complexes. We have compared MHC and peptide recognition by panels of CTL lines specific for the Tax and M1 peptides presented by HLA-A2 plus Tax and M1 peptide/HLA-A2-specific human Fabs that were selected from a naive phage display library. Collectively, the results indicate both striking similarities and important differences between Fab and TCR recognition of MHC and peptide components of the Tax and M1/HLA-A2 complexes. These findings suggest that these two classes of immunoreceptors have solved the problem of specific recognition of peptide/MHC complexes by nonidentical mechanisms. This conclusion is important in part because it indicates that Ab engineering approaches could produce second-generation Ab molecules that more closely mimic TCR fine specificity. Such efforts may produce more efficacious diagnostic and therapeutic agents.

MHC recognition by hapten-specific HLA-A2-restricted CD8+ CTL.

T cell recognition by peptide-specific alphabeta TCRs involves not only recognition of the peptide, but also recognition of multiple molecular features on the surface of the MHC molecule to which the peptide has been bound. We have previously shown that TCRs that are specific for five different peptides presented by HLA-A2 recognize similar molecular features on the surface of the alpha1 and alpha2 helices of the HLA-A2 molecule. We next asked whether these same molecular features of the HLA-A2 molecule would be recognized by hapten-specific HLA-A2-restricted TCRs, given that hapten-specific T cells frequently show reduced MHC dependence/restriction. The results show that a panel of CD8+ CTL that are specific for the hapten DNP bound to two different peptides presented by HLA-A2 do the following: 1) show stringent MHC restriction, and 2) are largely affected by the same mutations on the HLA-A2 molecule that affected recognition by peptide-specific CTL. A small subset of this panel of CD8+ CTL can recognize a mutant HLA-A2 molecule in the absence of hapten. These data suggest that TCR recognition of a divergent repertoire of ligands presented by HLA-A2 is largely dependent upon common structural elements in the central portion of the peptide-binding site.

Cytokine gene expression and protein production in peripheral blood mononuclear cells of children with acute dengue virus infections.

Plasma leakage in dengue hemorrhagic fever (DHF) is associated with elevated plasma levels of cytokines. To define further the contribution of immune activation to DHF and the source of cytokines, we analyzed the production of cytokines in peripheral blood mononuclear cells (PBMC) obtained from children with dengue, using RT-PCR and immunostaining. Tumor necrosis factor-alpha (TNF-alpha) and TNF-beta expression was detected in all samples by PCR and in < 50% of samples by immunostaining. Interferon-gamma (IFN-gamma) expression was detected in < 50% of samples by either method. Interleukin-2 (IL-2) and IL-4 expression was detected in a few samples by immunostaining but was not detectable by PCR. We found greater expression of TNF-alpha and IL-4 in DHF than in dengue fever or other (non-dengue) febrile illnesses. These results support the model of immunopathogenesis of DHF. However, low levels of cytokine expression in PBMC suggest that cellular activation in tissues may contribute to high serum cytokine levels in DHF.