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Reports on a study that indicates evidence for the effects of a superantigen in rheumatoid arthritis. Background; Study details; Results; Conclusions.

The clinical development of cancer drugs is rapidly moving from empirical "one drug fits all" or development-by-tumor-type approaches towards more personalized treatment models. A deeper understanding of cancer and the immune system, novel technologies, and powerful analytics have fueled an increase in precision oncology approaches integrating the molecular profiles of the tumor with the clinical profile of the patient. While this approach has been successful for targeted therapies, the complex mode of action of immunotherapies will likely require integration of clinical profiling with more comprehensive profiling of the tumor, of the tumor microenvironment, and of the immune system of the patient. Integration of precision oncology into clinical research for immunotherapies is viewed as a means to better select patients in the early clinical phase of drug development to (1) maximize the benefit-to-risk ratio for the patient, (2) generate early proof of concept and proof of relevance for the investigational drug, and (3) inform on how to best combine or sequence the therapeutic with other drugs. Here we discuss the upsides and challenges of incorporating precision immuno-oncology into early-phase clinical trials.

Respiratory syncytial virus (RSV) causes significant disease in elderly adults, but an effective vaccine is not yet available. We have previously reported that vaccines consisting of engineered respiratory syncytial virus soluble fusion protein (RSV sF) adjuvanted with glucopyranosyl lipid A (GLA) in an oil-in-water emulsion (stable emulsion [SE]) induce RSV F-specific T and B cell responses in mice and rats that protect from viral challenge. Here, we evaluated the immunogenicity of GLA-SE adjuvanted RSV sF vs unadjuvanted RSV sF vaccines in cynomolgus macaques (Macaca fascicularis). RSV F-specific IgG, RSV neutralizing antibodies, and RSV F-specific T cell IFNγ ELISPOT responses induced by GLA-SE adjuvanted RSV sF peaked at week 6 at significantly higher levels than achieved by unadjuvanted RSV sF and remained detectable through week 24, demonstrating response longevity. Two weeks after a week 24 booster immunization, humoral and cellular responses reached levels similar to those seen at the earlier peak response. Importantly, the GLA-SE adjuvanted RSV sF vaccine induced cross-neutralizing antibodies to other RSV A and B strains as well as F-specific IgA and IgG memory B cells. GLA-SE adjuvanted RSV sF was also demonstrated to drive a Th1-biased response characterized by more IFNγ than IL-4. This study indicates that a GLA-SE adjuvanted RSV sF vaccine induces robust humoral and Th1-biased cellular immunity in non-human primates and may benefit human populations at risk for RSV disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Chemical protein synthesis is important for dissecting the molecular basis of protein function. Here we advance its scope by demonstrating the significant improvement of the multifaceted pharmaceutical profile of small proteins exclusively via a chemical-based approach. The focus of this work centered on CCL-5 (RANTES) derivatives with potent anti-HIV activity. The overall chemical strategy involved a combination of coded and noncoded amino acid mutagenesis, peptide backbone engineering, and site-specific polymer attachment. The ability to alter specific protein residues, as well as precise control of the position and type of polymer attachment, allows for the exploration of specific molecular designs and resulted in novel CCL-5 analogues with significant differences in their respective biochemical and pharmaceutical properties. Using this approach, the complex-interplay of variables contributing to the noncovalent self-association (aggregation) state, CCR-5 specificity, in vivo elimination half-life, and anti-HIV activity of CCL-5-based protein analogues could be empirically evaluated via total chemical synthesis. This work has led to the identification of potent (sub-nanomolar) anti-HIV proteins with significantly improved pharmaceutical profiles, and illustrates the increasing value of protein chemical synthesis in contemporary therapeutic discovery. These antiviral molecules provide a novel mechanism of action for the development of a new generation of anti-HIV therapeutics which are still desperately needed.

A synthetic strategy that allows for the site-specific attachment of polymers such as poly(ethylene glycol) (PEG) to protein pharmaceuticals is described. PEG was attached to a 67-amino acid fully synthetic CCL-5 (RANTES) analogue at its GAG binding site both to reduce aggregation and to increase the circulating lifetime. Effective protection of an Aoaa chemoselective linker during peptide assembly, total chemical protein synthesis, and protein folding was achieved with an isopropylidene group. Mild deprotection of the resulting folded synthetic protein and subsequent polymer attachment occur without interference with the native folded structure and activity.

We initially evaluated in mice the ability of naked DNA encoding intracellular forms of the E1E2 envelope proteins from HCV to induce antibody responses and compared the responses induced with the same plasmid adsorbed onto cationic poly (lactide co-glycolide) (PLG) microparticles. Although naked DNA was only able to induce detectable responses at the 100 microg dose level, making this approach impractical for evaluation in larger animals, PLG/DNA induced detectable responses at 10 microg. In addition, the PLG/DNA microparticles induced significantly enhanced responses to naked DNA when compared at the same dose level. Remarkably, PLG/DNA induced comparable responses to recombinant E1E2 protein adjuvanted with the emulsion MF59. Furthermore, PLG/DNA effectively primed for a booster response with protein immunization, while naked DNA did not. Therefore, PLG/DNA was selected for further evaluation in a non-human primate model. In a study in rhesus macaques, PLG/DNA induced seroconversion in 3/3 animals following three immunizations. Although the antibody responses appeared lower than those induced with recombinant protein adjuvanted with MF59, following a fourth dose, PLG/DNA and protein induced comparable responses. However, a single booster dose of recombinant protein administered to the animals previously immunized with PLG/DNA induced much higher responses. In addition, one of three animals immunized with PLG/DNA showed a cytotoxic T lymphocyte response in peripheral blood lymphocytes. In conclusion, cationic PLG microparticles with adsorbed HCV DNA generates potent immune responses.

In this study, we evaluated the potency of a human papillomavirus (HPV) virus-like particle (VLP)-based vaccine at generating HPV type 11 (HPV-11)-specific cellular and humoral immune responses in seronegative women. The vaccine was administered by intramuscular immunizations at months 0, 2, and 6. A fourth immunization was administered to approximately half of the women at month 12. All vaccine recipients had positive HPV-11 VLP-specific lymphoproliferative responses at month 3 following the second immunization (geometric mean lymphoproliferative stimulation index [SI] = 28.4; 95% confidence interval [CI] = 16.9 to 48.0) and HPV-11 VLP-specific antibody titers following the first immunization at month 1 (geometric mean antibody titer = 53.9 milli-Merck units/ml, 95% CI, 34.8 to 83.7). In contrast, lymphoproliferative and antibody titer responses were never detected in the participants who received placebo. Relatively homogeneous lymphoproliferative responses were observed in all vaccinated women. The mean lymphoproliferative SI of the vaccinated group over the first 12 months of the study was 7.6-fold greater than that of the placebo group following the initial immunization. The cellular immune responses generated by VLP immunization were both Th1 and Th2, since peripheral blood mononuclear cells from vaccinees, but not placebo recipients, secreted interleukin 2 (IL-2), IL-5, and gamma interferon (IFN-gamma) in response to in vitro stimulation with HPV-11 VLP. The proliferation-based SI was moderately correlated with IFN-gamma production and significantly correlated with IL-2 production after the third immunization (P = 0.078 and 0.002, respectively). The robust lymphoproliferative responses were specific for HPV-11, since SIs generated against bovine papillomavirus and HPV-16 VLPs were not generally observed and when detected were similar pre- and postimmunization.

Naturally occurring hepatitis C virus (HCV) infection has long been thought to induce a weak immunity which is insufficient to protect an individual from subsequent infections and has cast doubt on the ability to develop effective vaccines. A series of intrahepatic genetic inoculations (IHGI) with type 1a HCV RNA were performed in a chimpanzee to determine whether a form of genetic immunization might stimulate protective immunity. We demonstrate that the chimpanzee not only developed protective immunity to the homologous type 1a RNA after rechallenge by IHGI but was also protected from chronic HCV infection after sequential rechallenge with 100 50% chimpanzee infectious doses of a heterologous type 1a (H77) and 1b (HC-J4) whole-virus inoculum. These results offer encouragement to pursue the development of HCV vaccines.

Loading of most endogenous peptides on major histocompatibility complex class I molecules is conditional on their transport into the endoplasmic reticulum (ER) by the peptide transporter TAP. We describe an HSV-2/1 cross-reactive cytotoxic T-cell (CTL) epitope that is processed in a TAP1-independent manner in vivo following immunization of TAP1-/- mice with naked DNA or a recombinant vaccinia virus. These data indicated that TAP1-independent processing of endogenous proteins is sufficient to prime CTLs in vivo. TAP1-independent processing of this epitope was not due to ER targeting by signal sequences and exogenous loading of MHC-I molecules and was not influenced by the amino acids flanking this epitope. In contrast, TAP1-/- mice infected with HSV-2 or HSV-2 mutants did not mount a CTL response against this epitope., (Copyright 2001 Academic Press.)

Current therapies for the treatment of hepatitis C virus (HCV) infection are only effective in a restricted number of patients. Cellular immune responses, particularly those mediated by CD8(+) CTLs, are thought to play a role in the control of infection and the response to antiviral therapies. Because the Core protein is the most conserved HCV protein among genotypes, we evaluated the ability of a Core prototype vaccine to prime cellular immune responses in rhesus macaques. Since there are serious concerns about using a genetic vaccine encoding for Core, this vaccine was a nonclassical ISCOM formulation in which the Core protein was adsorbed onto (not entrapped within) the ISCOMATRIX, resulting in approximately 1-microm particulates (as opposed to 40 nm for classical ISCOM formulations). We report that this Core-ISCOM prototype vaccine primed strong CD4(+) and CD8(+) T cell responses. Using intracellular staining for cytokines, we show that in immunized animals 0.30-0.71 and 0.32-2.21% of the circulating CD8(+) and CD4(+) T cells, respectively, were specific for naturally processed HCV Core peptides. Furthermore, this vaccine elicited a Th0-type response and induced a high titer of Abs against Core and long-lived cellular immune responses. Finally, we provide evidence that Core-ISCOM could serve as an adjuvant for the HCV envelope protein E1E2. Thus, these data provide evidence that Core-ISCOM is effective at inducing cellular and humoral immune responses in nonhuman primates.

The immunology of hepatitis C virus (HCV) infection should be studied in the context of HCV antigen expression in the liver, because HCV primarily infects this organ. Indeed, the nature, function, and fate of T cells primed after antigen expression in the liver might differ from those primed when antigens are expressed systemically or in other organs, because the nature of the antigen-presenting cells (APCs) involved may be different. In addition, the normal liver contains a resident population of lymphocytes that differ from those present at other sites. Thus, we investigated whether HCV-specific CD8(+) cytotoxic T cells (CTLs) could be elicited following portal vein (PV) injection of plasmid DNA in mice whose hepatic veins were transiently occluded. We show that PV injection of mice with "naked" DNA expressing the HCV-NS5a protein, under the control of a liver-specific enhancer/promoter, resulted in NS5a expression in the liver and the priming of HCV-specific CTLs. These results suggested that such a model might be relevant to the study of HCV-specific immune responses primed during natural infection.

Priming of strong cellular immune responses to hepatitis C (HCV) is thought to be important for eradication of infection. Although productive infection of HCV occurs only reproducibly in humans and chimpanzees, definition of HCV-specific T cell epitopes in mice is necessary to screen efficiently HCV vaccine strategies for their ability to prime cellular immune responses. Out of seven strains of mice screened for immunodominant CTL epitopes against HCV-1a Core, E2, NS5a and NS5b, only one epitope (p214K9) in only one mouse strain was identified. Enumeration of p214K9-specific CD8+ cells by flow cytometry revealed that the number of epitope specific CTL primed by 'naked' DNA immunization was lower than that reported during viral infection. The p214K9 epitope described here, combined with analysis of CTL responses by flow cytometry, should be instrumental in ranking various HCV vaccine strategies for their ability to prime CTL responses.

Despite advances in the clinical management of HIV infection, using combinations of antiretroviral pharmaceuticals, a safe and efficacious vaccine is needed to limit the AIDS pandemic. It is now thought that an effective HIV-1 vaccine should prime both cross-neutralizing antibodies and long-lasting cytotoxic CD8+ T lymphocytes (CTLs) recognizing multiple codominant HIV-1 epitopes. To that end, many novel vaccine strategies have been tested. However, only a few of these strategies, beside those relying on live-attenuated viruses, are able to prime strong CTL responses in nonhuman primates and humans. In this study, three rhesus macaques were immunized with HIV-1 p55gag virus-like particles (VLPs) in the absence of adjuvant to assess the potential of such a vaccine to prime CTL responses. After intramuscular injection of p55gag VLP, all three animals mounted CTL responses against HIV-1 p55gag. Notably, these CTLs primed by vaccination recognized naturally processed peptides and were long lived (>8.5 months) both in the peripheral blood and draining lymph node. Furthermore, these CTLs were directed against multiple HIV-1 p55gag epitopes. This indicated that immunization with p55gag VLP primes strong MHC class I-restricted, CD8+ cell-mediated immune responses and suggested that HIV-1 p55gag VLPs should be a reasonable vaccine candidate, when combined with strategies priming cross-neutralizing antibodies.

Dendritic cells (DCs) are powerful antigen-presenting cells. Because DCs are rare cells, methods to produce them in vitro are valuable ways to study their biologic properties and to generate cells for immunotherapy. This study defines the antigen-presenting properties of DCs generated in vitro from CD34+ cells of patients with breast cancer. The combination of cytokines flt3 ligand + c-kit ligand + granulocyte-macrophage colony-stimulating factor (GM-CSF) + interleukin-4 (IL-4) + tumor necrosis factor-alpha (TNF-alpha) was used to maximize the output of mature DCs in the culture of CD34+ cells while minimizing the production of monocytes. Cells grew and differentiated into DCs as measured by a time-dependent upregulation of cell surface antigens major histocompatibility complex class II, CD1a, CD80, CD86, CD40, and CD4, so that 40% +/- 9% (n = 6) of cells in culture at day 15 were CD1a+CD14-. Markers were acquired in the same sequence as on monocytes induced to differentiate with GM-CSF + IL-4. Differentiation was marked by a time-dependent increase in allostimulatory function, which, at its peak, was more potent than in cultures of DCs generated from monocytes with GM-CSF + IL-4, but was comparable on a cell-to-cell basis to that of mature monocytes cultured in flt3-ligand + c-kit-ligand + GM-CSF + IL-4 + TNF-alpha. Both CD34+ cell-derived and monocyte-derived DCs were able to process and to present tetanus toxoid and keyhole limpet hemocyanin to autologous T cells and to present major histocompatibility class I-binding peptides to CD8+ cytotoxic T lymphocytes inducing interferon-gamma production. Altogether, these results suggest that DCs generated from CD34+ cells of patients with breast cancer with flt3 ligand, c-kit ligand, GM-CSF, IL-4, and TNF-alpha are competent antigen-presenting cells, particularly for CD8+ cytotoxic T lymphocytes, and resemble mature monocyte-derived DCs in the assays described here.

The TNF family is involved in the regulation of the immune system, and its members have been implicated in a variety of biological events such as apoptosis, cell proliferation, differentiation and survival. Here we present a new member of the TNF family, tumor necrosis factor superfamily member 20 (TNFSF20) that we have identified from the expressed sequence tag (EST) database and characterized. The human protein is a 285 amino acid long type II transmembrane protein and is 19% homologous to TNF in its extra-cellular domain. TNFSF20 is expressed at the surface of antigen presenting cells such as cells of the macrophagemonocyte lineage and dendritic cells. After treatment with bacterial lipopolysaccharide (LPS), TNFSF20 expression is downregulated at the surface of the expresssing cells, suggesting that the membrane-bound protein gets cleaved, and that a soluble factor is released in the extra-cellular compartment. The soluble form of the recombinant TNFSF20 induces proliferation of resting peripheral blood monocytes (PBMC) and cell death of activated lymphocytes. TNFSF20 might therefore play a critical role in the regulation of cell-mediated immune responses.

Vaccination can elicit CD8(+) cytotoxic T lymphocytes (CTL) that recognize peptides presented by class I MHC molecules. Relatively little is known, however, about the genetic factors that shape the repertoire of T cell clonotypes responding to any given epitope. We report here that H-2(b) mice immunized with a plasmid DNA vaccine or vaccinia virus encoding for HIV-1SF2p55gag elicit CD8(+) CTL against the H-2Db-restricted immunodominant epitope (pgagb). This response involved three different T cell populations based on their recognition of alloantigens: one that cross-reacted with the alloantigen H-2Ld, one that cross-reacted with H-2Kd, and one that did not cross-react with either H-2(d) or H-2(k) molecules. Using the TAP-deficient cell line T2-Ld, we showed that pgagb-specific CTL cross-react with H-2Ld and a yet unidentified self-peptide. In mice expressing H-2(b) and H-2(d) allotypes, we investigated whether tolerance to H-2(d) influenced the HIVp55gag-specific CTL repertoire as a consequence of thymic deletion of the cross-reactive CTL repertoire. In (H-2(dxb))F1 mice heterogygosity at the MHC-I level prevented maturation of some but not all TCR combinations specific for H-2Db+pgagb, illustrating the concept that self-tolerance can influence the repertoire of antiviral T cells., (Copyright 1998 Academic Press.)

Objective: To determine whether CD8+ cells inhibit HIV replication in vitro through the chemokines RANTES, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta., Design and Methods: CD8+ T-cell clones were screened for their ability to inhibit HIV-1SF33 replication in CD4+ cells using p24 antigen and HIV RNA levels as endpoints. It has been suggested that such inhibition is mediated by three type cc chemokines: RANTES, MIP-1 alpha and MIP-1 beta. To assess whether our T-cell clones inhibited HIV replication through a similar mechanism, the clones' ability to inhibit HIV-1SF33 replication was compared with their secretion of RANTES, MIP-1 alpha and MIP-1 beta. Moreover, we tested the effects of neutralizing antibodies (NAb) against these factors on the anti HIV-1SF33 activity of our clones as well as the direct effect of these recombinant cc-chemokines on HIV-1SF33 replication., Results: The CD8+ T-cell clone; tested differed by their capacity to inhibit HIV-1 replication. We showed no correlation between the ability of these clones to secrete RANTES, MIP-1 alpha and MIP-1 beta and their ability to repress HIV-1SF33 replication. In addition, this inhibitory activity against HIV-1SF33 could not be blocked by NAb directed against these chemokines, nor could these chemokines significantly inhibit HIV-1SF33 replication in acutely infected CD4+ cells in vitro., Conclusion: The data indicate that CD8+ cells can inhibit HIV-1SF33 replication in vitro by mechanisms that do not involve either cytotoxicity or RANTES, MIP-1 alpha and MIP-1 beta.

Interleukin (IL-4) can induce CD8 expression on mature CD4+ T cells. To study this phenomenon in more detail, we characterized CD8 expressed on IL-4-induced CD4+ CD8+ (double positive) T cell clones in comparison with that on CD8+ T cell clones. Using 2ST8-5H7 mAb that detects CD8 beta expression, we found that double positive T cell clones isolated with IL-4 express CD8 alpha but not beta, in contrast to CD8+ CTL cell clones, which express both chains of CD8. Northern blot analysis revealed that these double positive clones expressed CD8 alpha but not beta mRNA, indicating that CD8 alpha and beta are independently regulated at the pre-translational level. Immunoprecipitation experiments showed that CD8 expressed on a representative IL-4-induced double positive T cell clone consists mainly of homodimers of a single 34 kd protein of CD8 alpha. The amount of multimers detected from this clone was much less than that from a CD8+ CTL clone. These results suggest that persistent expression of CD8 beta is specific for the CD8+ lineage and may be involved in polymerization and stabilization of CD8 which enhances the efficiency of class I-restricted antigen recognition.

CD4 and CD8 antigens are simultaneously expressed on most of the cortical thymocytes, that weakly express the T-cell antigen receptor(TCR)/CD3 complex. Mature peripheral T cells, however, strongly express the TCR complex and are positive for either CD4 or CD8. Nevertheless, a small percentage of peripheral CD3+ T cells express CD4 and CD8 simultaneously. These mature, double positive cells could be intermediates between CD4+CD8+ thymocytes and mature, single positive T cells, or they may originate from single positive T cells that acquire either CD4 or CD8. Here we report that activation and culturing of cloned CD4+ T cells in interleukin-4 (IL-4), results in the acquisition of CD8 due to its de novo synthesis. The IL-4-induced co-expression of CD8 on CD4+ T cells is reversible, in that CD8 disappeared from double positive T-cell clones isolated in IL-4, when they were cultured in IL-2. CD8 induced by IL-4 can be functional as a monoclonal antibody to CD8 inhibited anti-CD3-mediated cytotoxicity by a double positive T-cell clone.

Human colostral secretory IgA (SIgA; predominantly present in dimeric of polymeric forms) induces receptors for the Fc portion of IgA (Fc alpha R) on cloned and noncloned human T cell lines. The binding of SIgA to its FcR was isotype specific, since it was not inhibited by IgG or IgM. Binding of SIgA was also not affected by ovalbumin asialoglycoprotein. In addition, SIgA blocked the binding of directly fluorescein isothiocyanate-labeled SIgA in a dose-dependent fashion, whereas IgG and IgM were ineffective, confirming the specificity of the binding. Expression of Fc alpha R was specifically induced by SIgA, whereas serum IgA (predominantly present in monomeric form) had no effect. In addition, IgG, IgM and IgE were ineffective. This induction of Fc alpha R by SIgA was dose dependent. Optimal induction was observed at concentrations of 500 micrograms/ml after incubation times of 48 h. Fc alpha R were predominantly induced on T cell lines and T cell clones derived from tonsils. T cell lines and T cell clones established from peripheral blood could only occasionally be induced to express Fc alpha R. Induction of Fc alpha R expression was obtained both with CD4+ and CD8+ T cell clones. Fc alpha R were readily induced on T cell clones tested up to 6 days after activation by alloantigen. T cell clones tested 10-12 days after alloantigen activation failed to respond to SIgA. These results indicate that the inducibility of Fc alpha R is related to the activation stage of the T cell clones.