Enzyme-linked Immunosorbent Spot Research Articles

A Degenerate HLA-DR Epitope Pool of HER-2/neu Reveals a NovelIn vivoImmunodominant Epitope, HER-2/neu88-102

Over the past two decades, there has been significant interest in targeting HER-2/neu in immune-based approaches for the treatment of HER-2/neu+ cancers. For example, peptide vaccination using a CD8 T cell-activating HER-2/neu epitope (amino acids 369-377) is an approach that is being considered in advanced phase clinical trials. Studies have suggested that the persistence of HER-2/neu-specific CD8 T cells could be improved by incorporating human leukocyte antigen (HLA) class II epitopes in the vaccine. Our goal in this study was to identify broad coverage HLA-DR epitopes of HER-2/neu, an antigen that is highly expressed in a variety of carcinomas. A combination of algorithms and HLA-DR-binding assays was used to identify HLA-DR epitopes of HER-2/neu antigen. Evidence of preexistent immunity in cancer patients against the identified epitopes was determined using IFN-gamma enzyme-linked immunosorbent spot (ELIspot) assay. Eighty-four HLA-DR epitopes of HER-2/neu were predicted, 15 of which had high binding affinity for > or =11 common HLA-DR molecules. A degenerate pool of four HLA-DR-restricted 15-amino acid epitopes (p59, p88, p422, and p885) was identified, against which >58% of breast and ovarian cancer patients had preexistent T-cell immunity. All four epitopes are naturally processed by antigen-presenting cells. Hardy-Weinberg analysis showed that the pool is useful in approximately 84% of population. Lastly, in this degenerate pool, we identified a novel in vivo immunodominant HLA-DR epitope, HER-2/neu(88-102) (p88). The broad coverage and natural immunity to this epitope pool suggests potential usefulness in HER-2/neu-targeting, immune-based therapies such as vaccines.

Persistent Expression of FLAG-tagged Micro dystrophin in Nonhuman Primates Following Intramuscular and Vascular Delivery

Animal models for Duchenne muscular dystrophy (DMD) have species limitations related to assessing function, immune response, and distribution of micro- or mini-dystrophins. Nonhuman primates (NHPs) provide the ideal model to optimize vector delivery across a vascular barrier and provide accurate dose estimates for widespread transduction. To address vascular delivery and dosing in rhesus macaques, we have generated a fusion construct that encodes an eight amino-acid FLAG epitope at the C-terminus of micro-dystrophin to facilitate translational studies targeting DMD. Intramuscular (IM) injection of AAV8.MCK.micro-dys.FLAG in the tibialis anterior (TA) of macaques demonstrated robust gene expression, with muscle transduction (50–79%) persisting for up to 5 months. Success by IM injection was followed by targeted vascular delivery studies using a fluoroscopy-guided catheter threaded through the femoral artery. Three months after gene transfer, >80% of muscle fibers showed gene expression in the targeted muscle. No cellular immune response to AAV8 capsid, micro-dystrophin, or the FLAG tag was detected by interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) at any time point with either route. In summary, an epitope-tagged micro-dystrophin cassette enhances the ability to evaluate site-specific localization and distribution of gene expression in the NHP in preparation for vascular delivery clinical trials.

Autoimmune Hemolytic Anemia Accompanied by Reactivation of an Epstein-Barr Virus Infection with Suppressed CTL Response to EBV-infected Cells in an Elderly Man

An 88-year-old man with autoimmune hemolytic anemia (AIHA) who had been treated with low dose prednisolone developed a sudden worsening of his anemia accompanied by reactivation of Epstein-Barr virus (EBV). We established EBV-infected spontaneous lymphoblastoid cell lines (LCL), performed an enzyme-linked immunosorbent spot assay, and confirmed a significantly suppressed EBV-specific cytotoxic T-cell (CTL) response to the LCL. EBV reactivation might have been brought about by suppressed CTL activity which could have been due to low dose PSL administration or aging. Since the EBV-DNA titer decreased as AIHA improved, we concluded that EBV might have played a role in the development of anemia.

Studies of the anti–platelet factor 4/heparin immune response: adapting the enzyme‐linked immunosorbent spot assay for detection of memory B cells against complex antigens

The anti-platelet factor 4 (PF4)/heparin immune response, which underlies heparin-induced thrombocytopenia (HIT), has several atypical features: relatively rapid onset even without previous heparin exposure, lack of immune anamnesis, and transience of antibody production. We modified the enzyme-linked immunosorbent spot (ELISPOT) assay to investigate for PF4/heparin-specific memory B cells in cardiac surgery patients, in whom the high anti-PF4/heparin immunization rate made a prospective study feasible. The PF4-containing antigen complexes were attached to microtiter plates via a spacer, rather than using nitrocellulose, and the final reaction enzyme substrate was added in melted agarose which, after rapid hardening, localized color development of enzyme-tagged anti-immunoglobulin G (IgG) probes to single PF4/heparin-specific B cells. This modified ELISPOT assay was applied to 58 consecutive patients (testing blood from preoperative baseline and Postoperative Days 6 and 10), in which we compared detectability of PF4/heparin-specific B cells to tetanus toxin-specific B cells (comparator group with presumed vaccination). No patient had detectable PF4/heparin-specific memory B cells at baseline. In 2 of 30 patients (6.7%) who formed anti-PF4/heparin IgG, PF4/heparin-specific memory B cells (three to four spots/well) were detected by Postoperative Day 10, whereas tetanus toxin-specific memory B cells were found in 12 of 24 (50.0%) patients tested (3-25 spots/well; p < 0.001). HIT lacks a strong memory B-cell response, perhaps explaining transience and lack of anamnesis of the anti-PF4/heparin immune response. The technical modifications we describe for the ELISPOT assay, which permit detection of B-cell reactions to complex antigens, could be useful for studying other immunohematologic disorders, for example, drug-dependent thrombocytopenia and acquired hemophilia.

Clinical and Immunologic Responses of HLA-A3 + Breast Cancer Patients Vaccinated with the HER2/ neu-Derived Peptide Vaccine, E75, in a Phase I/II Clinical Trial

We have treated disease-free breast cancer patients with an HER2/neu-derived peptide, E75, as an adjuvant vaccine. E75 was originally described as HLA-A2-restricted and has been previously tested in this population. Based on computer modeling, E75 is predicted to bind to HLA-A3, and preclinical data support this. We conducted a clinical trial of E75 in HLA-A3(+), A2(-) (A3) patients. Disease-free breast cancer patients were enrolled after standard therapy in phase I/II trials. A3 patients were enrolled in parallel with A2 patients and vaccinated with E75 and granulocyte-macrophage colony-stimulating factor immunoadjuvant. A2(-), A3(-) patients were followed as controls. Toxicities were graded. Immunologic responses were assessed by delayed-type hypersensitivity reactions and E75-specific interferon-gamma enzyme-linked immunosorbent spot assay. Clinical recurrences were documented. Thirteen A3 patients completed the vaccine schedule. Clinicopathologic features were similar between A3, A2, and control patients, except for more HER2/neu-overexpressing tumors in the A2 group and more estrogen-receptor/progesterone-receptor-negative tumors in A2 and A3 groups. Toxicity profiles and postvaccination delayed-type hypersensitivity were similar in A3 and A2 patients. Enzyme-linked immunosorbent spot assay results varied, but A3 patients' median spots increased pre- to postvaccination (p = 0.2). Recurrences were lower in the A3 group (7.7%) at 30-month median follow-up compared with published recurrence in A2-vaccinated (8.3%) and control groups (14.8%) at 26-month median follow-up. HLA restriction limits potential use of peptide-based cancer vaccines. This trial demonstrates that HLA-A3 patients respond similarly to E75 vaccination as HLA-A2 patients, suggesting the potential use of the E75 vaccine in up to 76% of the population.

T-Cell Immune Monitoring in Organ Transplantation

Chronic allograft injury remains the leading cause of graft loss despite improvements in immunosuppression, clinical risk stratification, and state-of-the-art antibody testing. Emerging results indicate that T-cell immune monitoring by cytokine enzyme-linked immunosorbent spot, as part of a comprehensive risk assessment platform, has the potential to guide decision making and improve outcomes after transplantation.

Timing of HAART Defines the Integrity of Memory B Cells and the Longevity of Humoral Responses in HIV-1 Vertically-Infected Children

Pensieroso S, Cagigi A, Palma P, et al. Proc Natl Acad Sci U S A. 2009;106(19):7939–7944PURPOSE OF THE STUDY. HIV infection induces a progressive decline in immune function that affects not only T-cell but also B-cell activities, including the progressive decline in memory B cells, markedly elevated serum immunoglobulin levels, impaired responsiveness to routine immunizations, and loss of specific antibodies previously generated. Treatment of adults with highly active antiretroviral therapy (HAART) reduces immunoglobulin levels and increases B-cell numbers. However, several studies indicate that the B-cell compartment does not recover completely and patients maintain impaired antibody responses to immunizations. The purpose of this study was to determine whether the timing of HAART initiation affected pediatric patients’ ability to generate and to maintain protective levels of antibodies to routine immunizations.STUDY POPULATION. Seventy children perinatally infected with HIV were studied with 50 healthy control subjects.METHODS. Patients and control subjects received childhood immunizations according to the national (Italian) vaccine protocol. Patients who started HAART within the first year of life were categorized as “early treated”; children treated after the first year of life were considered “late treated,” and this group was subdivided into individuals with controlled virus and those who developed virological failure. Peripheral blood cells were evaluated with standard flow cytometry for B-cell subsets, including memory B cells. Antigen-specific B-cell functions were measured with an enzyme-linked immunosorbent spot assay. Plasma antibody titers against measles, tetanus, and pneumococcal antigens were assayed with enzyme-linked immunosorbent assays.RESULTS. Early-treated patients maintained high percentages of memory B cells, compared with levels observed in healthy control subjects; patients who started HAART later showed lower percentages. Patients treated early maintained the capacity to generate antigen-specific memory B cells, and early HAART resulted in maintenance of multiple antibody levels above protective thresholds in HIV-infected children. Of concern, 25% of patients treated late failed to generate protective levels of antibodies to measles, and this number increased to >40% among those who experienced failure of antiretroviral therapy. In addition, >60% of subjects who received late HAART failed to maintain protective levels of antibodies to antigens including measles and tetanus; similar findings were noted in antibody responses to pneumococcal antigens.CONCLUSIONS. Early HAART is essential for maintenance of normal B-cell functions in perinatally HIV-infected children. Regardless of T-cell numbers and/or clinical status, the results of this study strongly indicate that newborns infected with HIV should be treated as early as possible to preserve immune functions.REVIEWER COMMENTS. Although T cells are critical for generating normal antibody responses, unique subsets of B lymphocytes are also essential in this regard. Specifically, marginal-zone B cells are required for the ability to generate polysaccharide-specific, “T-cell independent” antibodies. Infants <2 years of age are deficient in this subset of B lymphocytes. This study provides strong confirmation of many clinicians’ impressions that children who begin HAART at <1 year of age and who maintain viral suppression have normal immune functions by all measures that are clinically applied to the evaluation of this population. When to initiate HAART has been a long-term question among practitioners treating adults. Increasingly, earlier treatment (ie, at CD4+ cell counts of <500 cells per μL in adult patients) is being considered. The results of this study suggest that HAART should begin as soon as a diagnosis of HIV infection is made in perinatally exposed children.

Chemokine Receptor Blockade With a Synthetic Nonpeptide Compound Attenuates Cardiac Allograft Vasculopathy

BACKGROUND.: Recruitment of recipient mononuclear cells to the donor heart is a central event in the development of cardiac allograft vasculopathy (CAV). The role of individual chemokine receptors in this process is incompletely defined. TAK-779 (N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetra-hydro-2H-pyran-4-aminium chloride) is a synthetic nonpeptide antagonist of CCR5 and CXCR3. The purpose of this study is to determine if combined CCR5 and CXCR3 blockade by TAK-779 would attenuate CAV in an experimental model. METHODS.: We used a previously characterized murine model of CAV. Recipient mice were treated with TAK-779 or vehicle control. Donor hearts were harvested on day 24 posttransplantation and analyzed for intimal thickening and cellular infiltration. Recipient splenocytes were analyzed for proliferative response and interferon (IFN)-gamma production by enzyme-linked immunosorbent spot assay. The donor hearts were also examined for heme oxygenase-1 (HO-1) gene induction. RESULTS.: CCR5 and CXCR3 blockade by TAK-779 reduced the severity of intimal lesions (53+/-10% vs. 16+/-2%; P<0.05) and decreased the number of graft infiltrating CCR5 and CXCR3 CD4 and CD8 lymphocytes. Moreover, treatment with TAK-779 (a) decreased alloantigen-specific T-lymphocyte proliferation and number of IFN-gamma producing cells and (b) increased HO-1 gene transcript level in the allografts. CONCLUSIONS.: Antagonism of CCR5 and CXCR3 by TAK-779 is effective in controlling CAV. The beneficial effects of TAK-779 may be because of (a) reduction in CCR5 and CXCR3 T-lymphocyte subset infiltration into the graft, (b) attenuation of alloantigen-specific T-lymphocyte proliferative response and IFN-gamma production, and (c) induction of HO-1 gene. This compound may offer a novel approach in clinical management of CAV.

Coadministration of Telomerase Genetic Vaccine and a Novel TLR9 Agonist in Nonhuman Primates

The human telomerase reverse transcriptase (hTERT) is an attractive target for human cancer vaccination because its expression is reactivated in most human tumors. We have evaluated the ability of DNA electroporation (DNA-EP) and adenovirus serotype 6 (Ad6) to induce immune responses against hTERT in nonhuman primates (NHPs) (Macaca mulatta). Vaccination was effective in all treated animals, and the adaptive immune response remained detectable and long lasting without side effects. To further enhance the efficacy of the hTERT vaccine, we evaluated the combination of hTERT vaccine and a novel TLR9 agonist, referred to as immunomodulatory oligonucleotide (IMO). Monkeys were dosed weekly with IMO concurrently with the vaccine regimen and showed increases in cytokine secretion and activation of natural killer (NK) cells compared with the group that received vaccine alone. Using a peptide array, a specific profile of B-cell reactive epitopes was identified when hTERT vaccine was combined with IMO. The combination of IMO with hTERT genetic vaccine did not impact vaccine-induced TERT-specific cell-mediated immunity. Our results show that appropriate combination of a DNA-EP/Ad6-based cancer vaccine against hTERT with IMO induces multiple effects on innate and adaptive immune responses in NHPs.

Pretransplantation Cellular Alloreactivity Is Predictive of Acute Graft Rejection and 1-Year Graft Function in Kidney Transplant Recipients

To study cellular alloimmunity in kidney allograft recipients using an interferon-gamma enzyme-linked immunosorbent spot assay (ELISPOT). Donor splenocyte peripheral blood mononuclear cells were obtained during kidney recovery in 53 kidney recipients including 11 with positive panel-reactive antibodies pretransplantation. For ELISPOT data analysis, the spot number, size, and intensity were calculated, reflecting the volume of cytokine secretion at the single-cell level. Results were recalculated as the ratio of the values observed for donor-stimulated to unstimulated recipient cells corrected for residual donor activity. Significantly greater pretransplantation donor-stimulated activity was observed in recipients who experienced an acute rejection episode (ARE) within 1 year (P < .05). Mean change in spot number, size, and intensity in patients without or with AREs was 0.99 vs 3.33, 1.60 vs 6.05, and 1.40 vs 6.31, respectively. The assessed parameters were prognostic of high risk of ARE: 1.5-fold increase in spot number (ARE incidence, 52% vs 9%), 2.5-fold increase in spot size (ARE incidence, 53% vs 13%), and 2.7-fold increase in spot intensity (ARE incidence, 52% vs 9%). The 3 parameters correlated with 1-year serum creatinine concentration (P < .05). In 14 recipients, AREs could have been predicted in 11 using pretransplantation ELISPOT results, and in only 2 on the basis of panel-reactive antibodies. The ELISPOT-determined capacity of donor-induced reactivity observed in recipient cells obtained just before transplantation is predictive of risk of graft rejection and 1-year allograft function.

Identification of the murine AAVrh32.33 capsid-specific CD8+ T cell epitopes.

Adeno-associated virus (AAV) is an ideal gene therapy vector and is non-immunogenic in many small animal models. The stable gene expression commonly seen in murine models does not necessarily translate to nonhuman primates and higher-order species, highlighting the need for a better understanding of immune activation to these vectors. One capsid variant, AAVrh32.33, demonstrates a unique phenotype in murine muscle, reminiscent of what is often seen in higher-order species. AAVrh32.33 generates a strong CD8+ T-cell response to both capsid and encoded transgene antigens in a manner independent of transgene product or major histocompatability complex haplotype, making it an ideal candidate for studying immune activation to AAV in the mouse. To map the H-2b and H-2d dominant epitopes of the AAVrh32.33 capsid, C57BL/6 or Balb/C mice received an intramuscular injection of 1 x 10(11) genome copies of AAV2/rh32.33.CB.nLacZ. Three weeks later, splenocytes were harvested and stimulated in vitro with pooled or individual peptides from the AAVrh32.33 capsid peptide library and analysed by an interferon (IFN)-gamma enzyme-linked immunosorbent spot assay or intracellular cytokine staining. The immunodominant epitopes within the AAVrh32.33 capsid responsible for driving CD8+ T-cell responses to the capsid protein in C57BL/6 (SSYELPYVM) and Balb/C (KIPASGGNAL) mice were defined. Identification of dominant capsid epitopes will make it possible to monitor cellular responses to the AAV capsid in vivo, facilitating mechanistic studies critical to defining how cellular immunity to the AAV capsid arises and, ultimately, how the generation of capsid-specific T cells can be avoided to ensure safety in a gene therapy setting.

T-Cell Recognition of Prostatic Peptides in Men With Chronic Prostatitis/Chronic Pelvic Pain Syndrome

A potential etiology of chronic prostatitis/chronic pelvic pain syndrome is autoimmunity. We determined whether T cells from men with chronic prostatitis/chronic pelvic pain syndrome would recognize peptides derived from the normal self-prostatic proteins prostate specific antigen and prostatic acid phosphatase. CD4 T cells purified from peripheral blood of 31 patients with chronic prostatitis/chronic pelvic pain syndrome and from the buffy coat preparation of 27 normal male blood donors were stimulated in vitro with a panel of immunogenic peptides from prostate specific antigen and prostatic acid phosphatase, and assayed for reactivity with the peptides by interferon-gamma enzyme-linked immunosorbent spot assay. Intermediate resolution HLA typing was done by polymerase chain reaction. Peptides were also tested by binding assay against different class II alleles. Peptide PAP(173-192) was recognized more frequently by CD4 T cells from patients with chronic prostatitis/chronic pelvic pain syndrome than from healthy donors. The recognition of prostate specific antigen peptides was not statistically different when comparing cases to normal male blood donors individually. Peptide reactivity was more common in patients than in normal male blood donors for any prostate specific antigen peptide or any tested peptide. All peptides showed high promiscuity on binding assays. There was no association of cases with any specific HLA class II phenotype at intermediate resolution. CD4 T cells from patients with chronic prostatitis/chronic pelvic pain syndrome have a higher rate of recognizing the self-prostatic proteins prostatic acid phosphatase and prostate specific antigen compared to those from normal male blood donors. Data provide further evidence to support the role of autoimmunity in some men with chronic prostatitis/chronic pelvic pain syndrome.

A Study of the Specificity of Lymphocytes in Nevirapine-Induced Skin Rash

Nevirapine treatment can cause a skin rash. We developed an animal model of this rash and determined that the 12-hydroxylation metabolic pathway is responsible for the rash, and treatment of animals with 12-OH-nevirapine also leads to a rash. In the present study, we investigated the specificity of lymphocytes in nevirapine-induced skin rash. Brown Norway rats were treated with nevirapine or 12-OH-nevirapine to induce a rash. Lymph nodes were removed, and the response of lymphocytes to nevirapine and its metabolites/analogs was determined by cytokine production (enzyme-linked immunosorbent assay, enzyme-linked immunosorbent spot assay, and Luminex) and proliferation (alamar blue assay). Subsets of lymphocytes were depleted to determine which cells were responsible for cytokine production. Lymphocytes from animals rechallenged with nevirapine proliferated to nevirapine, but not to 12-OH-nevirapine or 4-chloro-nevirapine. They also produced interferon-gamma (IFN-gamma) when exposed to nevirapine, significantly less when exposed to 4-chloro-nevirapine, and very little when exposed to 12-OH-nevirapine, even though oxidation to 12-OH-nevirapine is required to induce the rash. Moreover, the specificity of lymphocytes from 12-OH-nevirapine-treated rats was the same, i.e., responding to nevirapine more than to 12-OH-nevirapine, even though these animals had never been exposed to nevirapine. A Luminex immunoassay showed that a variety of other cytokines/chemokines were also produced by nevirapine-stimulated lymphocytes. CD4(+) cells were the major source of IFN-gamma. The specificity of lymphocytes in activation assays cannot be used to determine what initiated an immune response. This has significant implications for understanding the evolution of an immune response and the basis of the pharmacological interaction hypothesis.

AAV-1–mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells

AbstractIn a clinical trial for adeno-associated virus serotype 1 (AAV-1)–mediated gene transfer to muscle for lipoprotein lipase (LPL) deficiency, 1 subject from the high-dose cohort experienced a transient increase in the muscle enzyme creatine phosphokinase (CPK) 4 weeks after gene transfer. Simultaneously, after an initial downward trend consistent with expression of LPL, plasma triglyceride levels returned to baseline. We characterized B- and T-cell responses to the vector and the transgene product in the subjects enrolled in this study. IFN-γ enzyme-linked immunosorbent spot (ELISpot) and intracellular cytokine staining assays performed on peripheral blood mononuclear cells (PBMCs) from the subject who experienced the CPK elevation showed the activation of capsid-specific CD4+ and CD8+ T cells. Four of 8 subjects had detectable T-cell responses to capsid with dose-dependent kinetics of appearance. Subjects with detectable T-cell responses to capsid also had higher anti–AAV-1 IgG3 antibody titer. No subject developed B- or T-cell responses to the LPL transgene product. These findings suggest that T-cell responses directed to the AAV-1 capsid are dose-dependent. Whether they also limit the duration of expression of the transgene at higher doses is unclear, and will require additional analyses at later time points.

T Cell Memory Response to Pneumococcal Protein Antigens in an Area of High Pneumococcal Carriage and Disease

Streptococcus pneumoniae is a leading cause of vaccine-preventable disease worldwide. Pneumococcal protein antigens are currently under study as components of potential vaccines that offer protection against multiple serotypes. We have therefore characterized T cell pneumococcal immunity acquired through asymptomatic carriage. Peripheral blood mononuclear cells from 40 healthy Gambian adults were stimulated with supernatants derived from S. pneumoniae strain (D39), 2 isogenic mutant strains lacking either pneumolysin or choline binding protein A, and recombinant pneumolysin. Immune responses were measured by cellular proliferation and by interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot and bioplex cytokine assays. Nasopharyngeal swabs were cultured to determine carriage rates. S. pneumoniae nasopharyngeal carriage was detected in 60% of individuals. Both effector and resting (or central) CD4(+) T cell memory were frequently present to a range of pneumococcal antigens. However, the level of the effector memory response did not relate to current nasopharyngeal carriage. Pneumolysin was not immunodominant in these T cell responses but induced a distinct proinflammatory profile (high IFN-gamma, IL-12[p40], and L-17 levels and low IL-10 and IL-13 levels). In this population, T cell-mediated immunological memory potentially capable of pathogen clearance and immune surveillance is common but is not associated with the absolute interruption of pneumococcal carriage. How this naturally acquired immune memory influences pneumococcal vaccine efficacy remains to be determined.

Concurrent Trastuzumab and HER2/neu-Specific Vaccination in Patients With Metastatic Breast Cancer

The primary objectives of this phase I/II study were to evaluate the safety and immunogenicity of combination therapy consisting of concurrent trastuzumab and human epidermal growth factor receptor 2 (HER2)/neu-specific vaccination in patients with HER2/neu-overexpressing metastatic breast cancer. Twenty-two patients with stage IV HER2/neu-positive breast cancer receiving trastuzumab therapy were vaccinated with an HER2/neu T-helper peptide-based vaccine. Toxicity was graded according to National Cancer Institute criteria, and antigen specific T-cell immunity was assessed by interferon gamma enzyme-linked immunosorbent spot assay. Data on progression-free and overall survival were collected. Concurrent trastuzumab and HER2/neu vaccinations were well tolerated, with 15% of patients experiencing an asymptomatic decline in left ventricular ejection fraction below the normal range during combination therapy. Although many patients had pre-existing immunity specific for HER2/neu and other breast cancer antigens while treated with trastuzumab alone, that immunity could be significantly boosted and maintained with vaccination. Epitope spreading within HER2/neu and to additional tumor-related proteins was stimulated by immunization, and the magnitude of the T-cell response generated was significantly inversely correlated with serum transforming growth factor beta levels. At a median follow-up of 36 months from the first vaccine, the median overall survival in the study population has not been reached. Combination therapy with trastuzumab and a HER2/neu vaccine is associated with minimal toxicity and results in prolonged, robust, antigen-specific immune responses in treated patients.

Persistent parvovirus B19 infection detected by specific CD4+ T‐cell responses in a patient with hepatitis and polyarthritis

We, here, report the case of a parvovirus B19 infection in an immunocompetent male patient presenting with acute hepatitis and polyarthritis. To follow the course of infection, we used a previously established enzyme-linked immunosorbent spot assay (ELISPOT) technique to detect CD4+ T cells specific for viral proteins. Even though symptoms of arthritis and hepatitis resolved in the immunocompetent individual within a few weeks, viral DNA in serum and CD4+ T cells specific for the viral protein VP1 unique region were still detectable more than 6 month after the onset of symptoms, thus pointing to a persistent state of infection. On the basis of this observation, we hypothesize that the intensity of liver involvement correlates with the likelihood of developing persistent parvovirus B19 infection. The described ELISPOT technique to detect virus-specific CD4+ T cells provides an excellent tool to analyse the state of parvovirus B19 infection for future studies to test this hypothesis.

Immunize and disappear—Safety-optimized mRNA vaccination with a panel of 29 allergens

The spread of type I allergic diseases has reached epidemic dimensions. The success of therapeutic intervention is limited, and hence prophylactic vaccination is now seriously considered. However, immunization of healthy individuals requires safety standards far beyond those applicable for therapeutic approaches. mRNAs encoding allergen molecules represent an attractive tool for preventive vaccination because of the inherent safety features of this vaccine type. In the current study we investigated whether mRNA constructs would be capable of protecting against type I allergic reactions in a murine model using the grass pollen allergen Phl p 5 and 28 other major pollen, food, animal, mold, and latex allergens. BALB/c mice were immunized intradermally either with conventional or replicase-based mRNA constructs. Subsequently, animals were sensitized by means of subcutaneous injection of allergen/alum, followed by airway provocation. IgG1/IgG2a/IgE titers were determined by using ELISAs. Allergen-specific functional IgE levels were assessed by using the basophil release assay. Measurement of cytokines in splenocyte cultures and bronchoalveolar lavage fluids were performed by using enzyme-linked immunosorbent spot assays/sandwich ELISAs. Eosinophil and CD8(+) counts in bronchoalveolar lavage specimens were determined by means of flow cytometry. Airway hyperreactivity was assessed with whole-body plethysmography and invasive resistance/dynamic compliance measurement. mRNA vaccination proved its antiallergic efficacy in terms of IgG subclass distribution, functional IgE suppression, reduction of IL-4 and IL-5 levels, induction of IFN-gamma-producing cells, and reduction of airway hyperreactivity and eosinophil counts in the lung. Immunization with mRNA induces T(H)1-biased immune responses similar to those elicited through DNA-based vaccination but additionally offers the advantage of a superior safety profile.

DNA/Amphiphilic Block Copolymer Nanospheres Promote Low-dose DNA Vaccination

Intramuscular (i.m.) DNA vaccination induces strong cellular immune responses in the mouse, but only at DNA doses that cannot be achieved in humans. Because antigen expression is weak after naked DNA injection, we screened five nonionic block copolymers of poly(ethyleneoxide)-poly(propyleneoxide) (PEO-PPO) for their ability to enhance DNA vaccination using a beta-galactosidase (betaGal) encoding plasmid, pCMV-betaGal, as immunogen. At a high DNA dose, formulation with the tetrafunctional block copolymers 304 (molecular weight [MW] 1,650) and 704 (MW 5,500) and the triblock copolymer Lutrol (MW 8,600) increased betaGal-specific interferon-gamma enzyme-linked immunosorbent spot (ELISPOT) responses 2-2.5-fold. More importantly, 704 allowed significant reductions in the dose of antigen-encoding plasmid. A single injection of 2 microg pCMV-betaGal with 704 gave humoral and ELISPOT responses equivalent to those obtained with 100 microg naked DNA and conferred protection in tumor vaccination models. However, 704 had no adjuvant properties for betaGal protein, and immune responses were only elicited by low doses of pCMV-betaGal formulated with 704 if noncoding carrier DNA was added to maintain total DNA dose at 20 microg. Overall, these results show that formulation with 704 and carrier DNA can reduce the dose of antigen-encoding plasmid by at least 50-fold.

Enhancement of anti-tumor immunity by high levels of Th1 and Th17 with a combination of dendritic cell fusion hybrids and regulatory T cell depletion in pancreatic cancer

Regulatory T cells (Tregs) play an important role in immunological self-tolerance and protect the host from autoimmune disease. However, in cancer immunity, Tregs might block anti-tumor immune responses. Therefore, the depletion of Tregs using a specific agent that suppresses its function or population, such as an anti-CD25 antibody, could promote anti-tumor immune responses. In the present study, a cytotoxicity assay, enzyme-linked immunosorbent spot (ELISPOT) assay and measuring cytokine secretion, were used to study the efficacy of Treg depletion by anti-CD25 antibody added to a dendritic cell/tumor cell (DC/TC) fusion hybrid vaccine in a murine pancreatic cancer model. All the mice treated with the combined therapy of fusion hybrid vaccine and Treg depletion rejected tumor growth in a challenging test, although the rejection rate was 20% both for mice that received the fusion hybrids alone or Treg depletion alone. In addition, combined therapy showed a significantly improved survival in comparison to other treatment or control groups. The NK cell activity for DC/TC fusion + Treg depletion was significantly higher than that for the other treatment groups. Cytotoxic T lymphocyte (CTL) activity for DC/TC could potentially be enhanced by the addition of Treg depletion therapy. The treatments including DC/TC fusion induced IFN-gamma secreting effector cells in ELISPOT assays. Furthermore, a cytometric beads array assay used to measure cytokine secretion showed that DC/TC fusion + Treg depletion stimulated the highest levels of IFN-gamma Th1/Th2 ratios and Th17. These results demonstrate that Treg depletion combined with DC/TC fusion hybrid vaccine enhanced the efficacy of immunotherapy in pancreatic cancer by activating CTLs and NK cells.