The effect of antigen dose on T cell-targeting vaccine outcome.

During the past 3-4 decades, an increasing amount of evidence has pointed to the complex role of the antigen dose or T cell receptor (TCR) stimulation strength on the subsequent type, duration and "flavor" or quality of the response. Antigen dose was initially shown to impact Th1/Th2 bias, and later also shown to differentially affect development and induction of Tregs, Th17, T-follicular helper (Tfh), cells, and others. In recent years the quality of both CD4/8 T cells during infections, cancer and/or autoimmunity has turned out to be critical for subsequent disease outcome. Importantly, different vaccination strategies also lead to different types of T cell responses, and the role of the antigen dose is emerging as an important factor as well as a tool for investigators to utilize in fine-tuning vaccine efficacy. This commentary will highlight essential background of how antigen dose can impact and affect the quality of T cell responses, and discuss how this translates in different vaccine settings.

Effects of Cross-Presentation, Antigen Processing, and Peptide Binding in HIV Evasion of T Cell Immunity.

Unlike cytosolic processing and presentation of viral Ags by virus-infected cells, Ags first expressed in infected nonprofessional APCs, such as CD4+ T cells in the case of HIV, are taken up by dendritic cells and cross-presented. This generally requires entry through the endocytic pathway, where endosomal proteases have first access for processing. Thus, understanding virus escape during cross-presentation requires an understanding of resistance to endosomal proteases, such as cathepsin S (CatS). We have modified HIV-1MN gp120 by mutating a key CatS cleavage site (Thr322Thr323) in the V3 loop of the immunodominant epitope IGPGRAFYTT to IGPGRAFYVV to prevent digestion. We found this mutation to facilitate cross-presentation and provide evidence from MHC binding and X-ray crystallographic structural studies that this results from preservation of the epitope rather than an increased epitope affinity for the MHC class I molecule. In contrast, when the protein is expressed by a vaccinia virus in the cytosol, the wild-type protein is immunogenic without this mutation. These proof-of-concept results show that a virus like HIV, infecting predominantly nonprofessional presenting cells, can escape T cell recognition by incorporating a CatS cleavage site that leads to destruction of an immunodominant epitope when the Ag undergoes endosomal cross-presentation.

Paradoxical myeloid-derived suppressor cell reduction in the bone marrow of SIV chronically infected macaques.

Myeloid derived suppressor cells (MDSCs), which suppress anti-tumor or anti-viral immune responses, are expanded in the peripheral blood and tissues of patients/animals with cancer or viral infectious diseases. We here show that in chronic SIV infection of Indian rhesus macaques, the frequency of MDSCs in the bone marrow (BM) was paradoxically and unexpectedly decreased, but increased in peripheral blood. Reduction of BM MDSCs was found in both CD14+MDSC and Lin-CD15+MDSC subsets. The reduction of MDSCs correlated with high plasma viral loads and low CD4+ T cell counts, suggesting that depletion of BM MDSCs was associated with SIV/AIDS disease progression. Of note, in SHIVSF162P4-infected macaques, which naturally control viral replication within a few months of infection, the frequency of MDSCs in the bone marrow was unchanged. To investigate the mechanisms by which BM MDSCs were reduced during chronic SIV infection, we tested several hypotheses: depletion due to viral infection, alterations in MDSC trafficking, and/or poor MDSC replenishment. We found that the possible mobilization of MDSCs from BM to peripheral tissues and the slow self-replenishment of MDSCs in the BM, along with the viral infection-induced depletion, all contribute to the observed BM MDSC reduction. We first demonstrate MDSC SIV infection in vivo. Correlation between BM CD14+MDSC reduction and CD8+ T cell activation in tissues is consistent with decreased immune suppression by MDSCs. Thus, depletion of BM MDSCs may contribute to the pathologic immune activation during chronic SIV infection and by extension HIV infection.

Low Antigen Dose in Adjuvant-Based Vaccination Selectively Induces CD4 T Cells with Enhanced Functional Avidity and Protective Efficacy.

T cells with high functional avidity can sense and respond to low levels of cognate Ag, a characteristic that is associated with more potent responses against tumors and many infections, including HIV. Although an important determinant of T cell efficacy, it has proven difficult to selectively induce T cells of high functional avidity through vaccination. Attempts to induce high-avidity T cells by low-dose in vivo vaccination failed because this strategy simply gave no response. Instead, selective induction of high-avidity T cells has required in vitro culturing of specific T cells with low Ag concentrations. In this study, we combined low vaccine Ag doses with a novel potent cationic liposomal adjuvant, cationic adjuvant formulation 09, consisting of dimethyldioctadecylammonium liposomes incorporating two immunomodulators (monomycolyl glycerol analog and polyinosinic-polycytidylic acid) that efficiently induces CD4 Th cells, as well as cross-primes CD8 CTL responses. We show that vaccination with low Ag dose selectively primes CD4 T cells of higher functional avidity, whereas CD8 T cell functional avidity was unrelated to vaccine dose in mice. Importantly, CD4 T cells of higher functional avidity induced by low-dose vaccinations showed higher cytokine release per cell and lower inhibitory receptor expression (PD-1, CTLA-4, and the apoptosis-inducing Fas death receptor) compared with their lower-avidity CD4 counterparts. Notably, increased functional CD4 T cell avidity improved antiviral efficacy of CD8 T cells. These data suggest that potent adjuvants, such as cationic adjuvant formulation 09, render low-dose vaccination a feasible and promising approach for generating high-avidity T cells through vaccination.

Lack of the programmed death-1 receptor renders host susceptible to enteric microbial infection through impairing the production of the mucosal natural killer cell effector molecules.

The programmed death-1 receptor is expressed on a wide range of immune effector cells, including T cells, natural killer T cells, dendritic cells, macrophages, and natural killer cells. In malignancies and chronic viral infections, increased expression of programmed death-1 by T cells is generally associated with a poor prognosis. However, its role in early host microbial defense at the intestinal mucosa is not well understood. We report that programmed death-1 expression is increased on conventional natural killer cells but not on CD4(+), CD8(+) or natural killer T cells, or CD11b(+) or CD11c(+) macrophages or dendritic cells after infection with the mouse pathogen Citrobacter rodentium. Mice genetically deficient in programmed death-1 or treated with anti-programmed death-1 antibody were more susceptible to acute enteric and systemic infection with Citrobacter rodentium. Wild-type but not programmed death-1-deficient mice infected with Citrobacter rodentium showed significantly increased expression of the conventional mucosal NK cell effector molecules granzyme B and perforin. In contrast, natural killer cells from programmed death-1-deficient mice had impaired expression of those mediators. Consistent with programmed death-1 being important for intracellular expression of natural killer cell effector molecules, mice depleted of natural killer cells and perforin-deficient mice manifested increased susceptibility to acute enteric infection with Citrobacter rodentium. Our findings suggest that increased programmed death-1 signaling pathway expression by conventional natural killer cells promotes host protection at the intestinal mucosa during acute infection with a bacterial gut pathogen by enhancing the expression and production of important effectors of natural killer cell function.

Comparing adjuvanted H28 and modified vaccinia virus ankara expressingH28 in a mouse and a non-human primate tuberculosis model.

Here we report for the first time on the immunogenicity and protective efficacy of a vaccine strategy involving the adjuvanted fusion protein "H28" (consisting of Ag85B-TB10.4-Rv2660c) and Modified Vaccinia Virus Ankara expressing H28. We show that a heterologous prime-boost regimen involving priming with H28 in a Th1 adjuvant followed by boosting with H28 expressed by MVA (H28/MVA28) induced the highest percentage of IFN-γ expressing T cells, the highest production of IFN-γ per single cell and the highest induction of CD8 T cells compared to either of the vaccines given alone. In contrast, in mice vaccinated with adjuvanted recombinant H28 alone (H28/H28) we observed the highest production of IL-2 per single cell and the highest frequency of antigen specific TNF-α/IL-2 expressing CD4 T cells pre and post infection. Interestingly, TNF-α/IL-2 expressing central memory-like CD4 T cells showed a significant positive correlation with protection at week 6 post infection, whereas the opposite was observed for post infection CD4 T cells producing only IFN-γ. Moreover, as a BCG booster vaccine in a clinically relevant non-human primate TB model, the H28/H28 vaccine strategy induced a slightly more prominent reduction of clinical disease and pathology for up to one year post infection compared to H28/MVA28. Taken together, our data showed that the adjuvanted subunit and MVA strategies led to different T cell subset combinations pre and post infection and that TNF-α/IL-2 double producing but not IFN-γ single producing CD4 T cell subsets correlated with protection in the mouse TB model. Moreover, our data demonstrated that the H28 vaccine antigen was able to induce strong protection in both a mouse and a non-human primate TB model.

Difference in TB10.4 T-cell epitope recognition following immunization with recombinant TB10.4, BCG or infection with Mycobacterium tuberculosis.

Most novel vaccines against infectious diseases are based on recombinant Ag; however, only few studies have compared Ag-specific immune responses induced by natural infection with that induced by the same Ag in a recombinant form. Here, we studied the epitope recognition pattern of the tuberculosis vaccine Ag, TB10.4, in a recombinant form, or when expressed by the pathogen Mycobacterium tuberculosis (M.tb), or by the current anti-tuberculosis vaccine, Mycobacterium bovis BCG. We showed that BCG and M.tb induced a similar CD4+ T-cell specific TB10.4 epitope-pattern, which differed completely from that induced by recombinant TB10.4. This difference was not due to post-translational modifications of TB10.4 or because TB10.4 is secreted from BCG and M.tb as a complex with Rv0287. In addition, BCG and TB10.4/CAF01 were both taken up by DC and macrophages in vivo, and in vitro uptake experiments revealed that both TB10.4 and BCG were transported to Lamp+-compartments. BCG and TB10.4 however, were directed to different types of Lamp+-compartments in the same APC, which may lead to different epitope recognition patterns. In conclusion, we show that different vectors can induce completely different recognition of the same protein.

CD4 and CD8 T cell responses to the M. tuberculosis Ag85B-TB10.4 promoted by adjuvanted subunit, adenovector or heterologous prime boost vaccination.

BACKGROUND: Although CD4 T cells are crucial for defense against M.tb, it is still not clear whether the optimal response against M.tb in fact involves both CD4 and CD8 T cells. To test this, we used a new vaccine strategy that generated a strong balanced T cell response consisting of both CD4 and CD8 T cells. METHODS AND FINDINGS: To compare CD4 and CD8 responses against Ag85B-TB10.4 (H4), H4 was delivered as a subunit vaccine in cationic liposomes (CAF01), expressed in Ad5 (Ad-H4) or as a heterologous prime boost vaccination. H4/CAF01 induced primarily CD4 T cells and Ad-H4 gave predominantly a CD8 T cell response. In contrast, the heterologous prime boost combination resulted in augmentation of both the CD4 and CD8 response. The majority (>40%) of the CD4 T cells induced by the heterologous prime boost protocol were polyfunctional, and expressed IFN-gamma(+), IL-2(+), and TNF-alpha(+), whereas most of the CD8 T cells expressed IFN-gamma(+) and TNF-alpha(+) and possessed strong cytotoxic potential. The heterologous prime boost protocol also gave an increase in protective efficacy against M.tb challenge compared to H4/CAF01 and Ad-H4. Both the H4 specific CD4 and CD8 T cells were recruited to the site of infection, at the onset of infection. However, compared to CD8 T cells, CD4 T cells showed more extensive recruitment and were the main T cell subset proliferating at the site of infection. CONCLUSIONS/SIGNIFICANCE: Heterologous prime boost based on H4, produced an additive effect on the priming of CD4 and CD8 cells and in terms of the protective capacity of the vaccine, and therefore represent an interesting new vaccine strategy against M.tb. However, CD4 and CD8 T cells respond very differently to live M.tb challenge, in a manner which supports the consensus that CD4 T cells do play the major role during the early stages of an M.tb infection.

Induction of CD8 T cells against a novel epitope in TB10.4: correlation with mycobacterial virulence and the presence of a functional region of difference-1.

Although infection with Mycobacterium tuberculosis (M.tb) induces a robust CD8 T cell response, the role of CD8 T cells in the defense against M.tb, and the mechanisms behind the induction of CD8 T cells, is still not clear. TB10.4 is a recently described Ag that is expressed by both bacillus Calmette-Guérin (BCG) and M.tb. In the present study, we describe a novel CD8 T cell epitope in TB10.4, TB10.4(3-11). We show that TB10.4(3-11)-specific CD8 T cells are induced at the onset of infection and are present throughout the infection in high numbers. TB10.4(3-11) CD8 T cells were recruited to the site of infection and expressed CD44, TNF-alpha, and IFN-gamma. In addition, TB10.4(3-11) CD8 T cells showed an up-regulation of FasL and LAMP-1/2 (CD107A/B), which correlated with a strong in vivo cytolytic activity. The induction of TB10.4(3-11)-specific CD8 T cells was less pronounced following infection with BCG compared to infection with M.tb. By using a rBCG expressing the genetic region of difference-1 (RD1), we show that the presence of a functional RD1 region increases the induction of TB10.4(3-11)-specific CD8 T cells as well as the bacterial virulence. Finally, as an M.tb variant lacking the genetic region RD1 also induced a significant amount of TB10.4(3-11)-specific CD8 T cells, and exhibited increased virulence compared with BCG, our data suggest that virulence in itself is also involved in generating a robust CD8 T cell response against mycobacterial epitopes, such as TB10.4(3-11).