Publications of Cornell University Medical College. Studies from the Departments of Pathology, Bacteriology and Immunology, Public Health & Preventive Medicine.

Subtitle varies.

New initiatives in immunology : [NIAID Study Group report].

"January 1981."

The immunology of rheumatism

Mode of access: Internet.

Fusion of interleukin-2 to subunit antigens increase their antigenicity in vitro due to an interleukin-2 receptor beta-mediated antigen uptake mechanism

Subunit vaccines, based on one or more epitopes, offer advantages over whole vaccines in terms of safety but are less antigenic. We investigated whether fusion of the cytokine interleukin-2 (IL-2) to influenza-derived subunit antigens could increase their antigenicity. The fusion of IL-2 to the subunit antigens increased their antigenicity in vitro. Encapsulation of the subunit antigen in liposomes also increased its antigenicity in vitro, yet encapsulation of the subunit IL-2 fusion did not. The use of anti-IL-2 receptor beta (IL-2Rbeta) antibody to block the receptor subunit on macrophages suggested that the adjuvancy exerted by IL-2 in our in vitro system is due to, at least in part, a previously unreported IL-2Rbeta-mediated antigen uptake mechanism.

Lipid and carbohydrate based adjuvant/carriers in immunology

This review discusses various issues regarding vaccines:what are they and how they work, safety aspects, the role of adjuvants and carriers in vaccination, synthetic peptides as immunogens, and new technologies for vaccine development and delivery including the identification of novel adjuvants for mucosal vaccine delivery. There has been a recent increase of interest, in the use of lipids and carbohydrates as adjuvants, and so a particular emphasis is placed on adjuvants derived from lipids or carbohydrates, or from both. Copyright (C) 2003 European Peptide Society and John Wiley Sons, Ltd.

Potential of lipid core peptide technology as a novel self-adjuvanting vaccine delivery system for multiple different synthetic peptide immunogens

This study demonstrates the effectiveness of a novel self-adjuvanting vaccine delivery system for multiple different synthetic peptide immunogens by use of lipid core peptide (LCP) technology. An LCP formulation incorporating two different protective epitopes of the surface antiphagocytic M protein of group A streptococci (GAS)-the causative agents of rheumatic fever and subsequent rheumatic heart disease-was tested in a murine parenteral immunization and GAS challenge model. Mice were immunized with the LCP-GAS formulation, which contains an M protein amino-terminal type-specific peptide sequence (8830) in combination with a conserved non-host-cross-reactive carboxy-terminal C-region peptide sequence (J8) of the M protein. Our data demonstrated immunogenicity of the LCP-8830-J8 formulation in B10.BR mice when coadministered in complete Freund's adjuvant and in the absence of a conventional adjuvant. In both cases, immunization led to induction of high-titer GAS peptide-specific serum immunoglobulin G antibody responses and induction of highly opsonic antibodies that did not cross-react with human heart tissue proteins. Moreover, mice were completely protected from GAS infection when immunized with LCP-8830-J8 in the presence or absence of a conventional adjuvant. Mice were not protected, however, following immunization with an LCP formulation containing a control peptide from a Schistosoma sp. These data support the potential of LCP technology in the development of novel self-adjuvanting multi-antigen component vaccines and point to the potential application of this system in the development of human vaccines against infectious diseases.

Durable complete clinical responses in a phase I/II trial using an autologous melanoma cell/dendritic cell vaccine

Advanced metastatic melanoma is incurable by standard treatments, but occasionally responds to immunotherapy. Recent trials using dendritic cells (DC) as a cellular adjuvant have concentrated on defined peptides as the source of antigens, and rely on foreign proteins as a source of help to generate a cell-mediated immune response. This approach limits patient accrual, because currently defined, non-mutated epitopes are restricted by a small number of human leucocyte antigens. It also fails to take advantage of mutated epitopes peculiar to the patient's own tumour, and of CD4(+) T lymphocytes as potential effectors of anti-tumour immunity. We therefore sought to determine whether a fully autologous DC vaccine is feasible, and of therapeutic benefit. Patients with American Joint Cancer Committee stage IV melanoma were treated with a fully autologous immunotherapy consisting of monocyte-derived DC, matured after culture with irradiated tumour cells. Of 19 patients enrolled into the trial, sufficient tumour was available to make treatments for 17. Of these, 12 received a complete priming phase of six cycles of either 0.9X10(6) or 5X10(6) DC/intradermal injection, at 2-weekly intervals. Where possible, treatment continued with the lower dose at 6-weekly intervals. The remaining five patients could not complete priming, due to progressive disease. Three of the 12 patients who completed priming have durable complete responses (average duration 3 5 months +), three had partial responses, and the remaining six had progressive disease (WHO criteria). Disease regression was not correlated with dose or with the development of delayed type hypersensitivity responses to intradermal challenge with irradiated, autologous tumour. However, plasma S-100B levels prior to the commencement of treatment correlated with objective clinical response (P = 0.05) and survival (log rank P < 0.001). The treatment had minimal side-effects and was well tolerated by all patients. Mature, monocyte-derived DC preparations exposed to appropriate tumour antigen sources can be reliably produced for patients with advanced metastatic melanoma, and in a subset of those patients with lower volume disease their repeated administration results in durable complete responses.

Fatty acids as haptens: exploring the limits of antigenicity

Fatty acids (FAs) are relatively small, hydrophobic and highly mobile molecular structures with vital biological functions and a ubiquitous distribution. Surprisingly, however, they can be rendered immunogenic. We have synthesised a novel immunogen in which dicarboxylic linoleic acid was conjugated to a carrier protein. Dicarboxylic fatty acids (DCA) differ from their normal counterparts only by their possession of a carboxyl group at each end of the molecule. When conjugated to proteins as haptens, they are, therefore, presented to the immune system with a free carboxyl group at the distal end, instead of a methyl group. Polyclonal IgG antibodies raised in response to this unique immunogen could bind not only conjugated hapten with high affinity, but also the equivalent free FA in mono and dicarboxylic form. Similar conjugates constructed from normal FAs produced much weaker antibody responses and could scarcely be considered antigenic at all. The cross-reactivities of the anti-DCA antibodies with FA variants differing in the number, position and configuration of their double bonds showed that the antibody paratope (binding site) was structured to accommodate the hapten in a way that depended on the precise shape of the acyl chain. We suggest that FAs become much more effective as B-cell epitopes when presented with their hydrophilic carboxyl group exposed on the surface of immunogenic conjugates. This type of epitope is determined by the particular double bond pattern of the unsaturated acyl chain, as well as the polar head group. (C) 2003 Elsevier Ltd. All rights reserved.

Monitoring dendritic cells in clinical practice using a new whole blood single-platform TruCOUNT assay

Dendritic cells (DC) from distinct DC subsets are essential contributors to normal human immune responses. Despite this, reliable assays that enable DC to be counted precisely have been slow to evolve. We have now developed a new single-platform flow cytometric assay based on TruCOUN(TM) beads and the whole blood Lyse/No-Wash protocol that allows precise counting of the CD14(-) blood DC subsets: CD11c(+)CD16(-) DC, CD11c(+)CD16(+) DC, CD123(hi) DC, CD1c(+) DC and BDCA-3(+) DC. This assay requires 50 mul of whole blood; does not rely on a hematology blood analyser for the absolute DC counts; allows DC counting in EDTA samples 24 It after collection; and is suitable for cord blood and peripheral blood. The data is highly reproducible with intra-assay and inter-assay coefficients of variation less than 3% and 11%, respectively. This assay does not produce the DC-T lymphocyte conjugates that result in DC counting abnormalities in conventional gradient-density separation procedures. Using the TruCOUNT assay, we established that absolute blood DC counts reduce with age in healthy individuals. In preliminary studies, we found a significantly lower absolute blood CD11c(+)CD16(+) DC count in stage III/IV versus stage I/II breast carcinoma patients and a lower absolute blood CD123(hi) DC count in multiple myeloma patients, compared to age-matched controls. These data indicate that scientific progress in DC counting technology will lead to the global standardization of DC counting and allow clinically meaningful data to be obtained. (C) 2003 Elsevier B.V. All rights reserved.

Cross-reactive recognition of human and primate cytomegalovirus sequences by human CD4 cytotoxic T lymphocytes specific for glycoprotein B and H

Although the importance of CD4(+) T cell responses to human cytonnegalovirus (HCMV) has recently been recognized in transplant and immunosuppressed patients, the precise specificity and nature of this response has remained largely unresolved. In the present study we have isolated CD4(+) CTL which recognize epitopes from HCMV glycoproteins gB and gH in association with two different HLA-DR antigens, DRA1*0101/DRB1*0701 (DR7) and DRA1*0101/DRB1*1101 (DR11). Comparison of amino acid sequences of HICMV isolates revealed that the gB and gH epitope sequences recognized by human CD4(+) T cells were not only conserved in clinical isolates from HCMV but also in CMV isolates from higher primates (chimpanzee, rhesus and baboon). Interestingly, these epitope sequences from chimpanzee, rhesus and baboon CMV are efficiently recognized by human CD4(+) CTL. More importantly, we show that gB-specific T cells from humans can also efficiently lyse pepticle-sensitized Patr-DR7(+) cells from chimpanzees. These findings suggest that conserved gB and gH epitopes should be considered while designing a prophylactic vaccine against HCMV. In addition, they also provide a functional basis for the conservation of MHC class 11 lineages between humans and Old World primates and open the possibility for the use of such primate models in vaccine development against HCMV.

Induction of Therapeutic T-Cell Responses to Subdominant Tumor-associated Viral Oncogene after Immunization with Replication-incompetent Polyepitope Adenovirus Vaccine

The EBV-encoded latent membrane proteins (LMP1 and LMP2), which are expressed in various EBV-associated malignancies have been proposed as a potential target for CTL-based therapy. However, the precursor frequency for LMP-specific CTL is generally low, and immunotherapy based on these antigens is often compromised by the poor immunogenicity and potential threat from their oncogenic potential. Here we have developed a replication-incompetent adenoviral vaccine that encodes multiple HLA class I-restricted CTL epitopes from LMP1 and LMP2 as a polyepitope. Immunization with this polyepitope vaccine consistently generated strong LMP-specific CTL responses in HLA A2/K-b mice, which can be readily detected by both ex vivo and in vivo T-cell assays. Furthermore, a human CTL response to LMP antigens can be rapidly expanded after stimulation with this recombinant polyepitope vector. These expanded T cells displayed strong lysis of autologous target cells sensitized with LMP1 and/or LMP2 CTL epitopes. More importantly, this adenoviral vaccine was also successfully used to reverse the outgrowth of LMP1-expressing tumors in HLA A2/K-b mice. These studies demonstrate that a replication-incompetent adenovirus polyepitope vaccine is an excellent tool for the induction of a protective CTL response directed toward multiple LMP CTL epitopes restricted through common HLA class I alleles prevalent in different ethnic groups where EBV-associated malignancies are endemic.

Herpesvirus-specific CD8 T cell immunity in old age: Cytomegalovirus impairs the response to a coresident EBV infection

Aging in humans is associated with increased infections and the reduced proliferative capacity of T cells, part of the more global phenomenon termed immune senescence. The etiology of immune senescence is unknown but the accumulation of virus-specific memory T cells may be a contributory factor. We have examined CD8 T cell responses to two persistent herpesvirus infections, CMV and EBV, and to a recurrent virus infection, influenza, in different age cohorts of healthy donors using HLA-peptide tetramers and intracellular cytokine detection. Of these, CMV appears to be the most immunogenic, with the CD8 T cell response representing over 10% of the CD8 pool in many elderly donors. Interestingly, the effect of age upon EBV-specific responses depends upon donor CMV sero-status. In CMV seropositive donors, the magnitude of the EBV-specific immune response is stable with age, but in CMV seronegative donors, the response to EBV increases significantly with age. By contrast, the influenza-specific CD8 T cell immune response decreases with age, independent of CMV status. The functional activity of the herpesvirus-specific immune response decreases in elderly donors, although the characteristic phenotypes of CMV- and EBV-specific memory populations are retained. This demonstrates that aging is associated with a marked accumulation of CMV-specific CD8 T cells together with a decrease in immediate effector function. Moreover, infection with CMV can reduce prevailing levels of immunity to EBV, another persistent virus. These results suggest that carriage of CMV may be detrimental to the immunocompetent host by suppressing heterologous virus-specific immunity during aging.

CCR5-Delta 32 mutation is strongly associated with primary sclerosing cholangitis

CCR5 plays a key role in the distribution of CD45RO+ T cells and contributes to generation of a T helper 1 immune response. CCR5-Delta32 is a 32-bp deletion associated with significant reduction in cell surface expression of the receptor. We investigated the role of CCR5-Delta32 on susceptibility to ulcerative colitis (UC), Crohn's disease ( CD) and primary sclerosing cholangitis (PSC). Genotype and allelic association analyses were performed in 162 patients with UC, 131 with CD, 71 with PSC and 419 matched controls. There was a significant difference in CCR5 genotype (OR 2.27, P = 0.003) between patients with sclerosing cholangitis and controls. Similarly, CCR5-Delta32 allele frequency was significantly higher in sclerosing cholangitis (17.6%) compared to controls (9.9%, OR 2.47, P = 0.007) and inflammatory bowel disease patients without sclerosing cholangitis ( 11.3%, OR 1.9, P = 0.027). There were no significant differences in CCR5 genotype or allele frequency between those with either UC or CD and controls. Genotypes with the CCR5-Delta32 variant were increased in patients with severe liver disease defined by portal hypertension and/or transplantation (45%) compared to those with mild liver disease (21%, OR 3.17, P = 0.03). The CCR5-Delta32 mutation may influence disease susceptibility and severity in patients with PSC.

Investigation of the immunocompetent cells that bind early pregnancy factor and preliminary studies of the early pregnancy factor target molecule

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties. It has been shown to suppress the delayed-type hypersensitivity response in mice as well as acute and chronic forms of experimental autommume encephalomyelitis in rats and mice, respectively. In previous studies, we have demonstrated that EPF binds to a population of lymphocytes and we hypothesized that it mediates its suppressive effects by binding to CD4(+) T cells. In the present study, we isolated monocytes and subpopulations of lymphocytes and labelled them with fluoresceinated EPF in order to determine which populations bind EPF. We demonstrated that EPF binds specifically to CD4(+), CD8(+), CD14(+) (monocytes) and CD56(+) NK cells but not to CD19(+) B cells. The identity of the molecule(s) on the cell surface that is targeted by EPF is unknown, but as EPF is an extracellular homologue of the intracellular protein chaperonin 10 (Cpn 10), we examined the possibility that the EPF receptor is a membrane-associated form of chaperonin 60 (Cpn60), the functional associate of Cpn 10 within the cell. The EPF target molecule on lymphocytes was visualized by chemical cross-linking of exogenous iodinated Cpn10 to cells and probed with anti-Cpn60. The effect of anti-Cpn60 on activity in the EPF bioassay, the rosette inhibition test, was also examined. In both instances, no specific interaction of this antibody and the putative receptor was observed. It was concluded that the cell surface molecule targeted by EPF is unlikely to be a homologue of Cpn60.