Susan J. Gagnon

Conversion of a T Cell Antagonist into an Agonist by Repairing a Defect in the TCR/Peptide/MHC Interface: Implications for TCR Signaling

The structure of the A6 alphabetaTCR/HTLV-1 Tax-peptide/MHC I complex with proline 6 of Tax substituted with alanine (P6A), an antagonist, is nearly identical to the structure with wild-type Tax agonist. Neither the proline in the agonist nor the alanine in the antagonist is contacted by the alphabetaTCR. Here, we demonstrate that antagonist activity of P6A is associated with low affinity of the A6 alphabetaTCR for Tax-P6A/HLA-A2. We show that stepwise repair of a packing defect in the TCR/MHC interface using N-alkylated amino acids results in stepwise increases in TCR affinity and activity. Kinetic and thermodynamic measurements suggest that for some ligands the range of T cell outcomes does not correlate with either their alphabetaTCR affinity or the half-life of the alphabetaTCR/peptide/MHC complex.

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.

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 vaccines 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.

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-15Rα, 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.

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

The α4 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 Crohns 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.

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.

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.

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

T cell recognition by peptide-specific αβ 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 α1 and α2 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.

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.

P19-50 LB. Role of vaccine-induced innate and adaptive immunity in controlling mucosal transmission of SIV in macaques

Background Mucosal immunity is critical in AIDS virus infection because most transmission is through mucosal surfaces and the GI mucosa is a major reservoir for virus replication. There is window in time in which mucosal T cells could potentially eradicate a nascent local mucosal infection before it disseminates. In most vaccine work, adjuvant-induced innate immunity was of interest only to improve adaptive immunity. Here, in addition to adaptive immunity, we also examine direct adjuvant effects on innate immune protection.