Assessment of vaccine-induced CD4 T cell responses to the 119-143 immunodominant region of the tumor-specific antigen NY-ESO-1 using DRB1*0101 tetramers.

Abstract: Purpose: NY-ESO-1 (ESO), a tumor-specific antigen of the cancer/testis group, is presently viewed as an important model antigen for the development of generic anticancer vaccines. The ESO119-143 region is immunodominant following immunization with a recombinant ESO vaccine. In this study, we generated DRB1*0101/ESO119-143 tetramers and used them to assess CD4 T-cell responses in vaccinated patients expressing DRB1*0101 (DR1). Experimental Design: We generated tetramers of DRB1*0101 incorporating peptide ESO119-143 using a previously described strategy. We assessed ESO119-143-specific CD4 T cells in peptide-stimulated post-vaccine cultures using the tetramers. We isolated DR1/ESO119-143 tetramer(+) cells by cell sorting and characterized them functionally. We assessed vaccine-induced CD4(+) DR1/ESO119-143 tetramer(+) T cells ex vivo and characterized them phenotypically. Results: Staining of cultures from vaccinated patients with DR1/ESO119-143 tetramers identified vaccine-induced CD4 T cells. Tetramer(+) cells isolated by cell sorting were of T(H)1 type and efficiently recognized full-length ESO. We identified ESO123-137 as the minimal optimal epitope recognized by DR1-restricted ESO-specific CD4 T cells. By assessing DR1/ESO119-143 tetramer(+) cells using T cell receptor (TCR) beta chain variable region (V beta)-specific antibodies, we identified several frequently used V beta. Finally, direct ex vivo staining of patients' CD4 T cells with tetramers allowed the direct quantification and phenotyping of vaccine-induced ESO-specific CD4 T cells. Conclusions: The development of DR1/ESO119-143 tetramers, allowing the direct visualization, isolation, and characterization of ESO-specific CD4 T cells, will be instrumental for the evaluation of spontaneous and vaccine-induced immune responses to this important tumor antigen in DR1-expressing patients

Immune response to hepatitis B vaccination in HIV-positive adults with isolated antibodies to HBV core antigen.

OBJECTIVE: To investigate the merits of vaccination against hepatitis B virus (HBV) in HIV-positive individuals with isolated antibodies to hepatitis B core antigen (anti-HBc). METHODS: HIV-positive patients with isolated anti-HBc and CD4 counts >200 cells/mm(3) received HBV vaccination. An antibody titre to hepatitis B surface antigen (anti-HBs titres) ≥10 IU/L one month post-vaccination was termed an anamnestic response; a titre <10 IU/L was termed a primary response. Patients with primary responses received a 3-dose vaccine course. Anti-HBs titres in all responders were measured 12 and 24 months post-vaccination. RESULTS: 37 patients were studied: 19 (51%) were co-infected with hepatitis C; median CD4 count was 443 cells/mm(3). 8/37 patients (22%) elicited an anamnestic response. 29/37 patients (78%) elicited a primary response. After a 3-dose vaccine course, 15/25 primary responders (60%) achieved anti-HBs titres ≥10 IU/L. HIV acquisition through injecting drug use was the only independent predictor of an anamnestic response (OR 22.9, CI 1.71-306.74, P=0.018). Median anti-HBs titres for anamnestic and primary responders were 51 IU/L (13-127) and 157 IU/L (25-650) respectively. Of all responders, 12/23 (52%) retained anti-HBs titres ≥10 IU/L at 24 months. Anti-HBs duration was not significantly different between anamnestic and primary responders. CONCLUSIONS: 23/37 HIV-positive patients (62%) with isolated anti-HBc achieved anti-HBs titres ≥10 IU/L after 1-3 vaccine doses. However, duration of this immune response was short-lived (

Phase I safety and immunogenicity trial of Plasmodium vivax CS derived long synthetic peptides adjuvanted with montanide ISA 720 or montanide ISA 51.

We assessed the safety, tolerability, and immunogenicity of a mixture of three synthetic peptides derived from the Plasmodium vivax circumsporozoite protein formulated in Montanide ISA 720 or Montanide ISA 51. Forty healthy malaria-naive volunteers were allocated to five experimental groups (A-E): four groups (A-D) were immunized intramuscularly with 50 and 100 μg/dose injections of a mixture of N, R, and C peptides formulated in the two different adjuvants at 0, 2, and 4 months and one group was administered placebo. Vaccines were immunogenic, safe, well tolerated, and no serious adverse events related to the vaccine occurred. Seroconversion occurred in > 90% of the vaccines and antibodies recognized the sporozoite protein on immunofluorescent antibody test. Vaccines in Montanide ISA 51 showed a higher sporozoite protein recognition and interferon production. Results encourage further testing of the vaccine protective efficacy.

Issuance of patient reminders for influenza vaccination by US-based primary care physicians during the first year of universal influenza vaccination recommendations.

To estimate the number of physician-reported influenza vaccination reminders during the 2010-2011 influenza season, the first influenza season after universal vaccination recommendations for influenza were introduced, we interviewed 493 members of the Physicians Consulting Network. Patient vaccination reminders are a highly effective means of increasing influenza vaccination; nonetheless, only one quarter of the primary care physicians interviewed issued influenza vaccination reminders during the first year of universal vaccination recommendations, highlighting the need to improve office-based promotion of influenza vaccination.

Global Genomic Diversity of Human Papillomavirus 6 Based on 724 Isolates and 190 Complete Genome Sequences.

Human papillomavirus type 6 (HPV6) is the major etiological agent of anogenital warts and laryngeal papillomas and has been included in both the quadrivalent and nonavalent prophylactic HPV vaccines. This study investigated the global genomic diversity of HPV6, using 724 isolates and 190 complete genomes from six continents, and the association of HPV6 genomic variants with geographical location, anatomical site of infection/disease, and gender. Initially, a 2,800-bp E5a-E5b-L1-LCR fragment was sequenced from 492/530 (92.8%) HPV6-positive samples collected for this study. Among them, 130 exhibited at least one single nucleotide polymorphism (SNP), indel, or amino acid change in the E5a-E5b-L1-LCR fragment and were sequenced in full. A global alignment and maximum likelihood tree of 190 complete HPV6 genomes (130 fully sequenced in this study and 60 obtained from sequence repositories) revealed two variant lineages, A and B, and five B sublineages: B1, B2, B3, B4, and B5. HPV6 (sub)lineage-specific SNPs and a 960-bp representative region for whole-genome-based phylogenetic clustering within the L2 open reading frame were identified. Multivariate logistic regression analysis revealed that lineage B predominated globally. Sublineage B3 was more common in Africa and North and South America, and lineage A was more common in Asia. Sublineages B1 and B3 were associated with anogenital infections, indicating a potential lesion-specific predilection of some HPV6 sublineages. Females had higher odds for infection with sublineage B3 than males. In conclusion, a global HPV6 phylogenetic analysis revealed the existence of two variant lineages and five sublineages, showing some degree of ethnogeographic, gender, and/or disease predilection in their distribution. IMPORTANCE: This study established the largest database of globally circulating HPV6 genomic variants and contributed a total of 130 new, complete HPV6 genome sequences to available sequence repositories. Two HPV6 variant lineages and five sublineages were identified and showed some degree of association with geographical location, anatomical site of infection/disease, and/or gender. We additionally identified several HPV6 lineage- and sublineage-specific SNPs to facilitate the identification of HPV6 variants and determined a representative region within the L2 gene that is suitable for HPV6 whole-genome-based phylogenetic analysis. This study complements and significantly expands the current knowledge of HPV6 genetic diversity and forms a comprehensive basis for future epidemiological, evolutionary, functional, pathogenicity, vaccination, and molecular assay development studies.

Development of an enzyme-linked immunosorbent assay for serodiagnosis of ringworm infection in cattle.

The aim of this study was to develop an in-house enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of ringworm infection in cattle. We used available recombinant forms of Trichophyton rubrum dipeptidyl peptidase V (TruDppV) and T. rubrum leucin aminopeptidase 2 (TruLap2), which are 98% identical to Trichophyton verrucosum orthologues. Field serum samples from 135 cattle with ringworm infection, as confirmed by direct microscopy, fluorescence microscopy, and PCR, and from 55 cattle without any apparent skin lesions or history of ringworm infection that served as negative controls were used. Sensitivities, specificities, and positive and negative predictive values were determined to evaluate the diagnostic value of our ELISA. Overall, the ELISAs based on recombinant TruDppV and TruLap2 discriminated well between infected animals and healthy controls. Highly significant differences (P < 0.0001, Mann-Whitney U test) were noted between optical density values obtained when sera from infected versus control cattle were tested. The ELISA developed for the detection of specific antibodies against DppV gave 89.6% sensitivity, 92.7% specificity, a 96.8% positive predictive value, and a 78.4% negative predictive value. The recombinant TruLap2-based ELISA displayed 88.1% sensitivity, 90.9% specificity, a 95.9% positive predictive value, and a 75.7% negative predictive value. To the best of our knowledge, this is the first ELISA based on recombinant antigens for assessing immune responses to ringworm infection in cattle; it is particularly suitable for epidemiological studies and also for the evaluation of vaccines and/or vaccination procedures.

Induction of potent antitumor CTL responses by recombinant vaccinia encoding a melan-A peptide analogue.

There is considerable interest in the development of vaccination strategies that would elicit strong tumor-specific CTL responses in cancer patients. One strategy consists of using recombinant viruses encoding amino acid sequences corresponding to natural CTL-defined peptide from tumor Ags as immunogens. However, studies with synthetic tumor antigenic peptides have demonstrated that introduction of single amino acid substitutions may dramatically increase their immunogenicity. In this study we have used a well-defined human melanoma tumor Ag system to test the possibility of translating the immunological potency of synthetic tumor antigenic peptide analogues into recombinant vaccinia viruses carrying constructs with the appropriate nucleotide substitutions. Our results indicate that the use of a mutated minigene construct directing the expression of a modified melanoma tumor Ag leads to improved Ag recognition and, more importantly, to enhanced immunogenicity. Thus, recombinant vaccinia viruses containing mutated minigene sequences may lead to new strategies for the induction of strong tumor-specific CTL responses in cancer patients.

Cancer immunotherapy drives implementation science in oncology.

Cancer immunotherapy has come a long way. The hope that immunological approaches may help cancer patients has sparked many initiatives in research and development (R&D). For many years, progress was modest and disappointments were frequent. Today, the increasing scientific and medical knowledge has established a solid basis for improvements. Considerable clinical success was first achieved for patients with hematological cancers. More recently, immunotherapy has entered center stage in the development of novel therapies against solid cancers. Together with R&D in angiogenesis, the field of immunology has fundamentally extended the scientific scope, which has evolved from a cancer-cell-centered view to a comprehensive and integrated vision of tumor biology. Current R&D is focused on a large array of possible disease mechanisms, driven by cancer cells, and amplified by tumor stroma, inflammatory and immunological actors, blood and lymph vessels, and the "macroenvironment," i.e. systemic mechanisms of the host, particularly of the haematopoietic system. Contrasting to this large spectrum of pathophysiological events promoting tumor growth, only a small number of biological mechanisms, namely of the immune system, have the potential to counteract tumor growth. They are of prime interest because therapeutic enhancement may result in clinical benefit for patients. This special issue is dedicated to immunotherapeutics against cancer, with particular emphasis on vaccination and combination therapies, providing updates and extended insight in this booming field.

Toll-like receptor 3 is necessary for dsRNA adjuvant effects.

Toll-like receptors (TLR) recognize pathogen associated molecular patterns, and the binding of their specific ligands triggers a proinflammatory response that helps to fight invading microorganisms, and can be harnessed to increase vaccine efficiency. The present study demonstrates that double-stranded RNA is a promising vaccine adjuvant able to increase both proliferation and activation of antigen-specific CD8(+) T cells. Importantly, TLR3 is required for this adjuvant effect, as TLR3 deficient recipients failed to enhance proliferation of adoptively transferred TCR transgenic CD8(+) T cells in the presence of double-stranded RNA. Finally, this study also shows that, in contrast to previous reports in humans, TLR3 does not exert direct costimulatory activity on CD8(+) T cells in mice.

The synthetic Plasmodium falciparum circumsporozoite peptide PfCS102 as a malaria vaccine candidate: a randomized controlled phase I trial.

BACKGROUND: Fully efficient vaccines against malaria pre-erythrocytic stage are still lacking. The objective of this dose/adjuvant-finding study was to evaluate the safety, reactogenicity and immunogenicity of a vaccine candidate based on a peptide spanning the C-terminal region of Plasmodium falciparum circumsporozoite protein (PfCS102) in malaria naive adults. METHODOLOGY AND PRINCIPAL FINDINGS: Thirty-six healthy malaria-naive adults were randomly distributed into three dose blocks (10, 30 and 100 microg) and vaccinated with PfCS102 in combination with either Montanide ISA 720 or GSK proprietary Adjuvant System AS02A at days 0, 60, and 180. Primary end-point (safety and reactogenicity) was based on the frequency of adverse events (AE) and of abnormal biological safety tests; secondary-end point (immunogenicity) on P. falciparum specific cell-mediated immunity and antibody response before and after immunization. The two adjuvant formulations were well tolerated and their safety profile was good. Most AEs were local and, when systemic, involved mainly fatigue and headache. Half the volunteers in AS02A groups experienced severe AEs (mainly erythema). After the third injection, 34 of 35 volunteers developed anti-PfCS102 and anti-sporozoite antibodies, and 28 of 35 demonstrated T-cell proliferative responses and IFN-gamma production. Five of 22 HLA-A2 and HLA-A3 volunteers displayed PfCS102 specific IFN-gamma secreting CD8(+) T cell responses. Responses were only marginally boosted after the 3(rd) vaccination and remained stable for 6 months. For both adjuvants, the dose of 10 microg was less immunogenic in comparison to 30 and 100 microg that induced similar responses. AS02A formulations with 30 microg or 100 microg PfCS102 induced about 10-folds higher antibody and IFN-gamma responses than Montanide formulations. CONCLUSIONS/SIGNIFICANCE: PfCS102 peptide was safe and highly immunogenic, allowing the design of more advanced trials to test its potential for protection. Two or three immunizations with a dose of 30 microg formulated with AS02A appeared the most appropriate choice for such studies. TRIAL REGISTRATION: Swissmedic.ch 2002 DR 1227.

Advances in vaccination against Helicobacter pylori.

A vaccination against Helicobacter pylori may represent both prophylactic and therapeutic approaches to the control of H. pylori infection. Different protective H. pylori-derived antigens, such as urease, vacuolating cytotoxin A, cytotoxin-associated antigen, neutrophil-activating protein and others can be produced at low cost in prokaryote expression systems and most of these antigens have already been administered to humans and shown to be safe. The recent development by Graham et al. of the model of H. pylori challenge in humans, the recent published clinical trials and the last insight generated in animal models of H. pylori infection regarding the immune mechanisms leading to vaccine-induced Helicobacter clearance will facilitate the evaluation of immunogenicity and efficacy of H. pylori vaccine candidates in Phase II and III clinical trials.

Prevalence rate and reasons for refusals of influenza vaccine in elderly.

More knowledge on the reasons for refusal of the influenza vaccine in elderly patients is essential to target groups for additional information, and hence improve coverage rate. The objective of the present study was to describe precisely the true motives for refusal. All patients aged over 64 who attended the Medical Outpatient Clinic, University of Lausanne, or their private practitioner's office during the 1999 and 2000 vaccination periods were included. Each patient was informed on influenza and its complications, as well as on the need for vaccination, its efficacy and adverse events. The vaccination was then proposed. In case of refusal, the reasons were investigated with an open question. Out of 1398 patients, 148 (12%) refused the vaccination. The main reasons for refusal were the perception of being in good health (16%), of not being susceptible to influenza (15%), of not having had the influenza vaccine in the past (15%), of having had a bad experience either personally or a relative (15%), and the uselessness of the vaccine (10%). Seventeen percent gave miscellaneous reasons and 12% no reason at all for refusal. Little epidemiological knowledge and resistance to change appear to be the major obstacles for wide acceptance of the vaccine by the elderly.

Serial protein labeling with infrared maleimide dyes to identify cysteine modifications

Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification and quantification of growing importance. However, saturation labeling of thiols with fluorescent dyes results in poor protein recuperation and therefore requires the use of large quantities of starting material. This is especially important in sequential dye-labeling steps when applied for an identification of cysteine modifications. First, we studied the effects of different detergents during labeling procedure, i.e. Tween 20, Triton X-100 and CHAPS, on protein yield and composition. Tween 20 and Triton X-100 resulted in yields of around 50% labeled proteins compared to only 10% with PBS alone and a most diversified 2-DE protein pattern. Secondly, Tween 20 was used for serial protein labeling with maleimid fluorophores, first to conjugate to accessible thiols and after a reduction to label with another fluorophore previously masked di-sulphide and/or oxidized proteins in frontal cortex autopsy tissue of a subject with mild Alzheimer's disease. Two-DE DIGE revealed a complex protein pattern of readily labeled thiols and di-sulphide and/or oxidized proteins. Seventeen proteins were identified by MALDI-TOF and by peptide fingerprints. Several proteins were oxidized and involved in Alzheimer's disease. However methionine oxidation was prevalent. Infrared DIGE may provide an additional tool for an identification of oxidation susceptible proteins.

Analysis of viral- and tumor antigen-specific CD4 T cell responses in healthy donors and melanoma patients

Résumé Des tentatives pour développer des traitements anti-cancéreux basés sur l'utilisation d'antigènes tumoraux ont commencé il y a plus de 10 ans. Depuis quelques années, un certain intérêt s'est portée sur une sous-population particulière des cellules du système immunitaire, les lymphocytes T CD4. Ces cellules jouent un rôle central dans les réponses immunitaires tant contre les virus que contre les cellules tumorales. Comme d'autres lymphocytes T, ces cellules sont activées de manière spécifique en reconnaissant un morceau d'antigène, appelé peptide. Ces peptides proviennent soit de protéines des cellules de l'hôte, soit des protéines étrangères (virus ou bactéries) soit de cellules transformées (cellules tumorales) et sont présentés aux lymphocytes T par des molécules du soi appelées CMH (complexe majeur d'histocompatibilité). Dans le cas des lymphocytes T CD4, ces molécules sont plus précisément des molécules du CMH de classe II (CMH II). Mis à part l'intérêt porté aux réponses médiées par les lymphocytes T cytotoxiques, un intérêt croissant pour les lymphocytes T CD4 s'est développé à cause de la place centrale qu'occupent ces cellules dans les réponses immunitaires. L'identification d'épitopes présentés par des molécules du CMH de classe II dérivés d'un grand nombre d'antigènes tumoraux, ainsi que le développement de techniques permettant de suivre les réponses immunitaires, offre des opportunités pour étudier de manière quantitative et qualitative les lymphocytes T CD4 spécifiques pour un antigène particulier chez des patients cancéreux. De plus, ces épitopes permettent d'induire des réponses médiées par les lymphocytes T CD4 et CD8 chez ces mêmes patients. Dans ce travail, notre premier but était de valider l'utilisation de multimères formés par des complexes peptide:molécules de CMH de class II (pCMH II) pour quantifier la réponse des cellules T CD4 dirigée contre l'épitope HA307-319 dérivé de la protéine hémaglutinine du virus de la grippe et présenté par HLA-DRB1*0401. En analysant des échantillons provenant de volontaires sains ayant reçus un vaccin contre la grippe, nous avons pu démontrer une expansion et une activation transitoires des lymphocytes T CD4 spécifiques pour le peptide HA307-319 après vaccination. De plus, les multimères pCMH II nous ont permis d'analyser plus en détails hétérogénéité des cellules T CD4 spécifiques pour le peptide HA307-319 présents dans le sang périphérique d'individus sains. Par la suite, notre but a été d'analyser les réponses des lymphocytes T CD4 spécifiques pour l'antigène Melan-A chez des patients atteints de mélanome métastatique. Nous avons tout d'abord démontré la présence de cellules T CD4 spécifiques pour l'épitope Melan-A51-73, présenté par HLA-DRBl*0401, qui avait déjà été préalablement décrit. Ensuite, nous avons décrit et caractérisé 2 nouveaux peptides issus de Melan-A qui sont présentés aux cellules T CD4 par différentes molécules du CMH de clans II. Des cellules spécifiques pour ces deux épitopes ont été trouvées chez 9/ 16 patients analysés. De plus, des multimères pCMH II chargés avec un des épitopes nous ont permis de détecter ex vivo des lymphocytes T CD4 spécifiques pour Melan-A dans le sang périphérique d'un patient atteint de mélanome. Mis ensemble, tous ces résultats suggèrent une potentielle utilisation des multimères pCMH II pour analyser en détail les lymphocytes T CD4 spécifiques d'antigènes définis. Cependant, le suivi ex vivo de telles cellules ne semble être possible que dans des cas bien particuliers. Néanmoins, les nouveaux épitopes issus de Melan-A et présentés par des molécules du CMH de classe II que nous avons décrits dans cette étude aideront à étudier plus en détails les lymphocytes T CD4 spécifiques pour Melan-A chez des patients atteints de mélanome, un sujet d'étude sur lequel peu de résultats sont à ce jour disponibles. Summary Attempts to develop cancer vaccines based on molecularly defined tumorassociated antigens were initiated more than 10 years ago. Apart from CTLmediated anti-tumor immunity, interests are. now focused on CD4 T cells that are central players of immune responses. The identification of MHC class-II-restricted epitopes from numerous tumor antigens together with the development of monitoring tools offers the opportunity to quantitatively and qualitatively study antigen-specific CD4 T lymphocytes in cancer patients and to induce both CTL and T helper responses in cancer patients. In this work, we first aimed at validating the use of peptide:MHC class II complex (pMHC II) multimers to quantitate the CD4 T cell response against the hemagglutinin-derived epitope HAso~-si9 from influenza virus presented by HLA-DRBl*0401. By analysing samples from healthy volunteers vaccinated with ananti-influenza vaccine, we could demonstrate a transient expansion and activation of HA-specific CD4 T cells after treatment. Moreover, pMHC II multimers helped us to study the heterogeneity of HAspecific CD4 T cells found in peripheral blood of healthy individuals. Then, we aimed to analyse Melan-A-specific CD4 T cell responses in metastatic melanoma patients. We first demonstrated the presence of CD4 T cells specific for the previously described Melan-A51_73 epitope presented by HLA-DRB 1 *0401 in peripheral blood of those patients. Second, we described and characterised 2 new Melan-A-derived peptides that are presented by different MHC II molecules to CD4 T cells. Specific cells for these epitopes were found in 9/ 16 rnelánoma patients analysed. In addition, pMHC II multimers loaded with one of the two epitopes allowed us to detect ex vivo Melan-A-specific CD4 T cells in peripheral blood of a melanoma patient. Together, these results suggest a potential use of pMHC II multimers in analysing in detail antigen-specific CD4 T cells. However, ex vivo monitoring of such cells will be possible only in particular conditions. Nevertheless, the new Melan-A-derived MHC II-restricted epitopes described here will help to study in more detail Melan-A-specific CD4 T cells in melanoma patients, a field where only scarce data are available.

Soluble MHC-peptide complexes containing long rigid linkers abolish CTL-mediated cytotoxicity.

Soluble MHC-peptide (pMHC) complexes induce intracellular calcium mobilization, diverse phosphorylation events, and death of CD8+ CTL, given that they are at least dimeric and co-engage CD8. By testing dimeric, tetrameric, and octameric pMHC complexes containing spacers of different lengths, we show that their ability to activate CTL decreases as the distance between their subunit MHC complexes increases. Remarkably, pMHC complexes containing long rigid polyproline spacers (&gt; or =80 A) inhibit target cell killing by cloned S14 CTL in a dose- and valence-dependent manner. Long octameric pMHC complexes abolished target cell lysis, even very strong lysis, at nanomolar concentrations. By contrast, an altered peptide ligand antagonist was only weakly inhibitory and only at high concentrations. Long D(b)-gp33 complexes strongly and specifically inhibited the D(b)-restricted lymphocytic choriomeningitis virus CTL response in vitro and in vivo. We show that complications related to transfer of peptide from soluble to cell-associated MHC molecules can be circumvented by using covalent pMHC complexes. Long pMHC complexes efficiently inhibited CTL target cell conjugate formation by interfering with TCR-mediated activation of LFA-1. Such reagents provide a new and powerful means to inhibit Ag-specific CTL responses and hence should be useful to blunt autoimmune disorders such as diabetes type I.