Induction Of Humoral Immune Responses Research Articles

Germinal Center B Cell and T Follicular Helper Cell Responses to Viral Vector and Protein-in-Adjuvant Vaccines.

There is great interest in the development of Ab-inducing subunit vaccines targeting infections, including HIV, malaria, and Ebola. We previously reported that adenovirus vectored vaccines are potent in priming Ab responses, but uncertainty remains regarding the optimal approach for induction of humoral immune responses. In this study, using OVA as a model Ag, we assessed the magnitude of the primary and anamnestic Ag–specific IgG responses of mice to four clinically relevant vaccine formulations: replication-deficient adenovirus; modified vaccinia Ankara (a poxvirus); protein with alum; and protein in the squalene oil-in-water adjuvant Addavax. We then used flow cytometric assays capable of measuring total and Ag-specific germinal center (GC) B cell and follicular Th cell responses to compare the induction of these responses by the different formulations. We report that adenovirus vectored vaccines induce Ag insert–specific GC B cell and Ab responses of a magnitude comparable to those induced by a potent protein/squalene oil-in-water formulation whereas—despite a robust overall GC response—the insert-specific GC B cell and Ab responses induced by modified vaccinia Ankara were extremely weak. Ag-specific follicular Th cell responses to adenovirus vectored vaccines exceeded those induced by other platforms at day 7 after immunization. We found little evidence that innate immune activation by adenovirus may act as an adjuvant in such a manner that the humoral response to a recombinant protein may be enhanced by coadministering with an adenovirus lacking a transgene of interest. Overall, these studies provide further support for the use of replication-deficient adenoviruses to induce humoral responses.

Mechanisms Underlying the Immune Response Generated by an Oral Vibrio cholerae Vaccine.

Mechanistic details underlying the resulting protective immune response generated by mucosal vaccines remain largely unknown. We investigated the involvement of Toll-like receptor signaling in the induction of humoral immune responses following oral immunization with Dukoral, comparing wild type mice with TLR-2-, TLR-4-, MyD88- and Trif-deficient mice. Although all groups generated similar levels of IgG antibodies, the proliferation of CD4+ T-cells in response to V. cholerae was shown to be mediated via MyD88/TLR signaling, and independently of Trif signaling. The results demonstrate differential requirements for generation of immune responses. These results also suggest that TLR pathways may be modulators of the quality of immune response elicited by the Dukoral vaccine. Determining the critical signaling pathways involved in the induction of immune response to this vaccine would be beneficial, and could contribute to more precisely-designed versions of other oral vaccines in the future.

A Plasmodium Promiscuous T Cell Epitope Delivered within the Ad5 Hexon Protein Enhances the Protective Efficacy of a Protein Based Malaria Vaccine.

A malaria vaccine is a public health priority. In order to produce an effective vaccine, a multistage approach targeting both the blood and the liver stage infection is desirable. The vaccine candidates also need to induce balanced immune responses including antibodies, CD4+ and CD8+ T cells. Protein-based subunit vaccines like RTS,S are able to induce strong antibody response but poor cellular reactivity. Adenoviral vectors have been effective inducing protective CD8+ T cell responses in several models including malaria; nonetheless this vaccine platform exhibits a limited induction of humoral immune responses. Two approaches have been used to improve the humoral immunogenicity of recombinant adenovirus vectors, the use of heterologous prime-boost regimens with recombinant proteins or the genetic modification of the hypervariable regions (HVR) of the capsid protein hexon to express B cell epitopes of interest. In this study, we describe the development of capsid modified Ad5 vectors that express a promiscuous Plasmodium yoelii T helper epitope denominated PyT53 within the hexon HVR2 region. Several regimens were tested in mice to determine the relevance of the hexon modification in enhancing protective immune responses induced by the previously described protein-based multi-stage experimental vaccine PyCMP. A heterologous prime-boost immunization regime that combines a hexon modified vector with transgenic expression of PyCMP followed by protein immunizations resulted in the induction of robust antibody and cellular immune responses in comparison to a similar regimen that includes a vector with unmodified hexon. These differences in immunogenicity translated into a better protective efficacy against both the hepatic and red blood cell stages of P. yoelii. To our knowledge, this is the first time that a hexon modification is used to deliver a promiscuous T cell epitope. Our data support the use of such modification to enhance the immunogenicity and protective efficacy of adenoviral based malaria vaccines.

Induction of humoral immune response against Pseudomonas aeruginosa flagellin(1-161) using gold nanoparticles as an adjuvant

Flagellin of Pseudomonas aeruginosa is an important vaccine candidate. N-terminal domains are highly conserved in both type a and type b flagellins. The efficacy of gold nanoparticles (AuNPs) conjugated to N-terminal domains of P. aeruginosa flagellin (flagellin(1-161)), as an immunogen in mice, has been assessed. The nanoparticles were conjugated to the recombinant protein through direct interaction of thiol molecules of the cysteines with AuNPs and formation of AuS bond. Flagellin(1-161), AuNP-flagellin(1-161), and flagellin(1-161) emulsified in Freund's adjuvant (FA: complete/incomplete Freund's adjuvant formulation) were administered subcutaneously to BALB/c mice. Mice given AuNP-flagellin(1-161) elicited high titers of anti-flagellin(1-161) antibodies compared with non-immune group and/or mice which received flagellin(1-161) without adjuvant. In whole cell ELISA, these antibodies effectively recognized the native flagellin on the bacteria. Opsonophagocytosis assay demonstrated the functional activity and specificity of anti-flagellin(1-161) antibodies raised by AuNP-flagellin(1-161) against homologous strain. All of the results were comparable with those obtained by use of FA. Taken together, this is the first report of conjugation of AuNPs to flagellin and evaluating its immune response against P. aeruginosa.

Incidence of Severe Malaria Syndromes and Status of Immune Responses among Khat Chewer Malaria Patients in Ethiopia.

Although more emphasis has been given to the genetic and environmental factors that determine host vulnerability to malaria, other factors that might have a crucial role in burdening the disease have not been evaluated yet. Therefore, this study was designed to assess the effect of khat chewing on the incidence of severe malaria syndromes and immune responses during malaria infection in an area where the two problems co-exist. Clinical, physical, demographic, hematological, biochemical and immunological data were collected from Plasmodium falciparum mono-infected malaria patients (age ≥ 10 years) seeking medication in Halaba Kulito and Jimma Health Centers. In addition, incidences of severe malaria symptoms were assessed. The data were analyzed using SPSS (version 20) software. Prevalence of current khat chewer malaria patients was 57.38% (95%CI =53-61.56%). Malaria symptoms such as hyperpyrexia, prostration and hyperparasitemia were significantly lower (P<0.05) among khat chewer malaria patients. However, relative risk to jaundice and renal failure were significantly higher (P<0.05) in khat chewers than in non-khat chewer malaria patients. Longer duration of khat use was positively associated with incidence of anemia. IgM and IgG antibody titers were significantly higher (P<0.05) among khat chewer malaria patients than among malaria positive non-chewers. Although levels of IgG subclasses in malaria patients did not show significant differences (P>0.05), IgG3 antibody was significantly higher (P<0.001) among khat chewer malaria patients. Moreover, IgM, IgG, IgG1and IgG3 antibodies had significant negative association (P<0.001) with parasite burden and clinical manifestations of severe malaria symptoms, but not with severe anemia and hypoglycemia. Additionally, a significant increment (P<0.05) in CD4+ T-lymphocyte population was observed among khat users. Khat might be an important risk factor for incidence of some severe malaria complications. Nevertheless, it can enhance induction of humoral immune response and CD4+ T-lymphocyte population during malaria infection. This calls for further investigation on the effect of khat on parasite or antigen-specifc protective malaria immunity and analysis of cytokines released upon malaria infection among khat chewers.

Entrapment in plasma microparticles: A promising strategy for antigen delivery

We report the preparation of plasma microparticles (PMPs) from autologous blood plasma for sustained in vivo delivery of the entrapped antigens. The PMPs were prepared by high speed-stirring of calcium-enriched plasma, mixed with the antigen to be entrapped, in mineral oil. The preparation of PMPs did not necessitate addition of any external protein/enzyme nor special laboratory setup. Our results suggest that the PMPs release the entrapped invertase in a sustained manner both in vitro and in vivo, especially after crosslinking with glutaraldehyde. The preparations are reasonably stable to proteolysis and constitute strong candidates for eliciting immune response. Induction of humoral immune response by the PMP-entrapped invertase, as evident from the high antibody titers, was remarkable and comparable with that observed in animals receiving the antigen emulsified with Freund's Complete Adjuvant. Isotypic analysis of antibodies showed a Th1-biased immune response in animals administered uncrosslinked or crosslinked PMPs-entrapped invertase, especially after a booster dose. The analysis in animals of the group immunized with adjuvant-emulsified antigen suggested a combined Th1 and Th2 response. PMP-entrapment also caused high expression of surface markers (CD80 and CD86) on antigen presenting cells, as well as effector T-cells surface markers (CD4(+) and CD8(+) ) as revealed by FACS. The study suggests that PMPs offer remarkable promise as adjuvant-free and biocompatible vaccine delivery systems.

Induction of immune response in broiler chickens immunized with recombinant FliC and challenged by Salmonella Typhimurium

The study examined (1) the immune response in broiler chickens after oral immunization with recombinant flagellin (rFliC) from Salmonella Typhimurium conjugated with sodium alginate microparticles, and the immune response enhancement in association with recombinant cholera toxin B subunit protein (rCTB) and pool of Lactobacillus spp. (PL). The immune responses were evaluated by dosage of IgY serum and IgA from intestinal fluid and immunostaining of CD8+ T lymphocytes in the cecum. The immunized animals were challenged with Salmonella Typhimurium (ST) 21 days after treatment. In all immunized groups, a significant increase (p&lt;0.05) was observed in IgA levels (μg/mL), especially three weeks after immunization. The serum IgY levels (μg/mL) were little affected by the treatments and differed significantly among groups only in the second post-immunization week (p&lt;0.05). After the challenge, the number of CD8+ T cells differed significantly between the treatments and negative control. Retrieval of Salmonella Typhimurium was not detected at 48 hours after the challenge in T2 (rFliC+rCTb), T3 (rFliC+PL) and T4 (rFliC+rCTB PL). The rFliC administered orally with or without rCTB and Lactobacillus spp. produces significant induction of humoral immune response, and the immunized chickens were more effective in eliminating Salmonella after challenge.

The induction of humoral immune responses to the orally administered Dukoral vaccine occurs independently of TLR signaling and DC maturation (P3188)

Abstract We investigated the involvement of TLR signaling in the induction of humoral and cell-mediated immune responses (CMIR) following oral and intramuscular (IM) immunization with Dukoral (killed whole-cell V. cholerae and cholera toxin B subunit, CTB) or its components by using TLR-2-, TLR-4-, MyD88- and Trif-deficient mice. We show that wild type and all groups of TLR-deficient mice generated similar levels of V. cholerae- and CTB-specific IgG1and IgG2c serum antibodies, fecal IgA antibodies and agglutinating antibodies, following oral immunization with Dukoral or CTB alone. However, IM immunization with Dukoral or CTB alone required MyD88 signaling for the induction of CTB-specific IgG1and IgG2c serum antibodies. In addition, our results show that CD4+ T-cell and CD19+ B-cell proliferation, maturation of DCs in response to vaccine components, as measured by CD80, CD86, CD40, and MHCII expression, and cytokine secretion including IFN-g occurred in a MyD88-dependent manner in response to V. cholerae or CTB stimulation. In contrast, in response to CTB stimulation, Trif negatively regulated both CD4+ T-cell and CD19+ B-cell proliferation. Overall, our results suggest that despite the impaired ability of MyD88-/- DCs to mature, humoral responses, following oral immunization with Dukoral, occurred independently of TLR signaling, while CMIR were TLR-dependent. However, TLR-independence is lost in the generation of humoral responses when the vaccine is administered parenterally.

Immunization strategies for Parkinson's disease

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Currently, no curative treatments or treatments that interdict disease progression are available. Over the past decade, immunization strategies were developed in our laboratories to combat disease progression. These strategies were developed in laboratory and animal models of human disease. Induction of humoral immune responses can be elicited against misfolded protein aggregates. Robust cell-mediated immunity against nitrated misfolded protein(s) accelerates disease progression through effector T cell responses that facilitate neuronal death. We propose that shifting the balance between effector and regulatory T cell activity can attenuate neurotoxic inflammatory events. We now summarize our works that support immune regulation in PD with the singular goal of restoring homeostatic glial responses. New methods to optimize immunization schemes and measure their clinical efficacy are discussed.

Induction of specific immune response and suppression of fertility by B-cell-epitope-based mimovirus vaccine

SPINLW1 (previously known as eppin (epididymal protease inhibitor)) is a target under intense scrutiny in the study of male contraceptive vaccines. B-cell-dominant epitopes are now recognized as key parts of the induction of humoral immune responses against target antigens. The generation of robust humoral responses in vivo has become a crucial problem in the development of modern vaccines. In this study, we developed a completely novel B-cell-dominant-epitope-based mimovirus vaccine, which is a kind of virus-size particulate antigen delivery system. The mimovirus successfully self-assembled from a cationic peptide containing a cell-penetrating peptide of TAT49-57 and a plasmid DNA encoding both three SPINLW1 (103-115) copies and adjuvant C3d3. The male mice were immunized with the epitope-based mimovirus vaccine, which resulted in a gradual elevation of specific serum IgG antibody levels. These reached a peak at week 4. Mating for the fertility assay showed that the mimovirus vaccine had accomplished a moderate fertility inhibition effect and investigation into the mechanism of action showed that it did so by interfering with the reproductive function of the sperm but that it did not damage the structures of the testes or cause serum testosterone to decline. Our results suggest an ideal protocol for suppressing fertility in mice by an engineered mimovirus vaccine.

PP-174 Comparison of Toxoplasma gondii excreted-secreted antigens from cell and cell free media in induction of humoral immune response

PP-174 Comparison of Toxoplasma gondii excreted-secreted antigens from cell and cell free media in induction of humoral immune response

Follicular dendritic cell activation during vaccination correlates with antibody response and germinal center formation (61.14)

Abstract FDCs are stromal cell of the lymphoid organs that play a central role in the induction of humoral immune response. FDCs deliver signal to B cells by trapping intact antigen onto their surface as antigen-antibodies or antigen-complement fragments immune complexes. Thus cognate B cells recognize the antigen presented by FDCs via BCR, while surface proteins or soluble factors expressed by FDCs serve as co-stimulation. FDC activation is pivotal for the origin of germinal center (GC) reaction, and TLR4 engagement appears critical for this purpose. Nevertheless, no studies analyzing the commitment of FDC during vaccination with licensed adjuvants have been performed. Here we show for the first time, that the intramuscular injection of adjuvants approved for human use leads to FDC activation, which occurs late after injection. In addition, we observe that the activation of FDCs upon injection of an adjuvanted antigen takes place only in the follicles where a GC reaction occurs and the number of follicles with activated FDCs strictly correlates with the antigen-specific antibody titers. Moreover, retention of the injected antigen is observed only in the follicles that contain activated FDCs. Thus the extent of the humoral immune response against an antigen correlates with the activation state of FDCs, which can be modulated by the presence of the adjuvants, maybe through an indirect effect. The elucidation of the adjuvant affect on FDCs can help to design more efficacious vaccines.

Induction of humoral immune response against PfMSP-1 19 and PvMSP-1 19 using gold nanoparticles along with alum

The C-terminal 19 kDa fragment of merozoite surface protein 1(MSP-1 19) is an important vaccine candidate but a poor immunogen with available human compatible adjuvants like alum. We have investigated if Gold Nanoparticles (GNPs) based delivery system would enhance immune response to recombinant PfMSP-1 19 and PvMSP-1 19. We found that PfMSP-1 19/PvMSP-1 19 coated on GNPs or in alum was poorly immunogenic in mice, a robust antibody response was observed when PfMSP-1 19/PvMSP-1 19 coated GNPs were formulated with alum. PfMSP-1 19/PvMSP-1 19 coated GNPs strongly recognized conformation specific monoclonal antibodies showing that capping on to GNPs did not compromise with conformational integrity of these antigens, which is critical for the generation of functional antibody response. Anti-PfMSP-1 19 antibodies raised with GNP-alum formulation recognized the native protein on parasite surface and showed invasion inhibition of Plasmodium falciparum parasite in an in vitro assay. Antigen coated GNPs with alum may be useful in the development of an alternate adjuvant formulation.

Administration of LPS-Stimulated Autologous Macrophages Induces α-Synuclein Aggregation in Dopaminergic Neurons of Rat Brain

Administration of autologous macrophages isolated from the abdominal cavity and stimulated in vitro with bacterial LPS to rats increased blood level of antibodies against α-synuclein. Antibody titer reached its maximum during week 5 of the experiment and exceeded the levels of anti-α-synuclein antibodies appeared in response to transplantation of non-stimulated macrophages. Brain immunohistochemistry showed that additional administration of LPS (250 μg/kg) to animals during week 4 after injection of LPS-stimulated macrophages led to α-synuclein accumulation in 9.4±3.2% dopaminergic neurons of the substantia nigra. These findings attest to induction of humoral immune response to α-synuclein in rats after administration of autologous LPS-stimulated macrophages, which can affect α-synuclein metabolism in dopaminergic neurons of the brain.

Follicular Dendritic Cell Makes Environmental Sense

Follicular dendritic cells (FDCs) are key organizers of B cell follicles and germinal centers. In this issue of Immunity, Suzuki et al. (2010) and Garin et al. (2010) identify the roles of Toll-like receptors in the responses of FDCs, providing a unique link between innate and adaptive immunity.

Abstract 3322: Protective antitumor immunity induced by ALDEFLUOR+ enriched cancer stem cells

Abstract The isolation of human cancer stem cells represents a new paradigm for the development of cancer treatments. In theory, therapies that expressly target cancer stem cells are more likely to be successful. So far, the majority of the stem cell studies have been confined to human tumors inoculated into severely immunosuppressed hosts (e.g. SCID mice), which are not suitable for immunologic evaluation. The lack of immunocompetent syngeneic animal tumor models where stem cells can be isolated has been the primary obstacle to evaluating the immunogenicity of tumor stem cells. In this study, we used ALDEFLUOR/ALDH1 as a marker and identified cancer stem cell (CSC)-enriched populations in two of our murine models (melanoma B16-D5 and pancreas cancer PAN-02). We found that: 1). a very small percentage (0.5-2.5%) of ALDEFLUOR+ cells exist in these murine tumors; 2). only sorted, and as few as 500 ALDEFLUOR+ cells could form tumors in syngeneic hosts; 3).the stem cell nature of the ALDELFUOR+ cells was confirmed by their ability to form spheres in culture and to self-renew in vivo. We hypothesize that CSCs may induce immunity specifically targeting CSCs. We proceeded to evaluate the protective antitumor responses induced by vaccination with dendritic cells (DC) pulsed with the lysate of ALDEFLUOR+ D5 cells (CSC-TPDC) in the syngeneic immunocompetent host. Normal mice were vaccinated with CSC-TPDC or DC pulsed with lysate from unsorted heterogeneous D5 cells (H-TPDC), followed by challenge with D5 tumor cells intravenously. Lungs were harvested 20 days later to enumerate metastases. Compared with non-vaccinated, PBS-injected controls, H-TPDC induced modest protective immunity against tumor challenge, which is consistent with our previous observations. More importantly, mice that received CSC-TPDC had significantly fewer lung metastases than both the control group and the H-TPDC treated group (p&amp;lt; 0.02). In a different setting utilizing CSC as antigen, CSC-TPDC vaccination induced a potent immune response that resulted in significant inhibition of s.c tumor growth. Like the pulmonary metastasis model, there was enhanced protection against tumor growth mediated by CSC-TPDC vs. H-TPDC. Splenocytes isolated from CSC-TPDC vaccinated hosts produced significantly higher levels of IgG and cytotoxic IgG2b. Together, these results suggest that enriched CSCs are immunogenic and are significantly more effective as an antigen source compared with unselected tumor cells in inducing protective antitumor immunity. CSC-conferred protection is associated with the induction of humoral immune responses. Establishing the superior immunogenicity of CSCs compared to unselected tumor cells in the immunocompetent host is novel because it has not been previously described. The findings from these studies provide a basis for developing novel immunological approaches for cancer treatment by specifically targeting cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3322.

Model α-mannoside conjugates: immunogenicity and induction of candidacidal activity

The effect of Candida cell wall mannan-derived alpha-oligomannoside structural components on the modulation of the immune system and their role in protective immunity are studied here. Semi-synthetic alpha-mannoside-bovine serum albumin conjugates were used for immunization of rabbits. Dimeric alpha-mannoside, representing Candida antigenic factor 1, was used as a model of linear alpha-mannoside, and pentameric alpha-mannoside was used as a model of branched oligomannoside side chain structure. The induction of humoral immune response and the functionality of the serum tested by induction of peripheral blood leukocyte (PBL) candidacidal activity are documented. Anti-Candida albicans serotype B immunoglobulins (IgG and IgM) levels were higher than anti-serotype A following immunization with both conjugates. Dimer-conjugate postimmunization sera evidently enhanced C. albicans killing activity of PBLs in candidacidal assay. The study shows the importance of alpha-mannoside structures in perspective anti-Candida vaccine with a broad spectrum of effectiveness.

Antibody-mediated protection and the mucosal immune system of the genital tract: relevance to vaccine design

Mucosal tissues of the genital tracts and the distal intestinal tract are portals of entry for infectious agents of sexually transmitted diseases, including HIV-1. Although the genital and intestinal tracts share a common embryologic origin and remain in anatomical proximity, these two sites display remarkably different immunologic features, including the levels, isotypes and molecular forms of immunoglobulins, and magnitudes and qualities of humoral and cellular immune responses. Thus, viral and bacterial infections of the genital tract or intravaginal immunizations induce, in the absence of mucosal adjuvants, minimal immune responses. Consequently, to induce relevant immune responses in the genital tract, alternative immunization routes have been explored, including systemic, intranasal, oral, or rectal immunization and their combinations. In limited studies performed in animals, systemic immunization with a subsequent mucosal (intranasal) immunization proved to be effective in the induction of humoral immune responses in genital tract secretions. The approaches have been explored to a limited extent in humans.

Fusion of HSP70 gene of Mycobacterium tuberculosis to hemagglutinin (H5) gene of avian influenza virus in DNA vaccine enhances its potency

A series of plasmids containing the HSP70 gene of Mycobacterium tuberculosis fused to the hemagglutinin (H5) gene of H5N1 avian influenza virus (AIV) (H5-HSP70 (heat shock protein 70) vaccine) or individual H5 gene (H5 vaccine) or HSP70 gene (HSP70 vaccine) were constructed based on the plasmid pcDNA3.1. Expression of H5 gene in Vero cells in vitro and in chickens in vivo was confirmed following their transfection and immunization with H5 or H5-HSP70 vaccines. Controls consisted of HSP70 vaccine, empty plasmid pcDNA3.1 and co-administered H5 and HSP70 vaccines. H5-HSP70 vaccine produced in chicken higher hemagglutination inhibition (HI) antibody titer than H5 vaccine. However, the increase was not statistically significant. We have demonstrated for the first time that the H5 DNA vaccine with fused HSP70 gene may produce an enhanced induction of humoral immune response to AIV in chickens.

Co-Existing Serological Immune Responses against RHAMM Might Be a Prerequisite for Strong Cellular Immune Responses of CD8-Positive T Cells in RHAMM-R3 Peptide Vaccination for Patients with Different Hematological Malignancies.

Abstract 3671Poster Board III-607For effective elimination of malignant cells by specific T cells a co-activation of CD4- and CD8-positive T cells might be important. We performed two RHAMM-R3 peptide vaccination trials using 300μg and 1000μg for patients with AML, MDS and multiple myeloma overexpressing RHAMM. Similar mild toxicity of both cohorts was found, only mild drug-related adverse events were observed such as erythema and induration of the skin. In the 300μg cohort we detected in 7/10 (70 %) patients specific immune responses and also positive clinical effects in 5/10 (50 %) patients. In the high dose peptide vaccination trial (1000μg peptide) 4/9 (44 %) patients showed positive immune responses. These patients showed an increase of CD8+RHAMM-R3 tetramer+/CD45RA+/CCR7-/CD27-/CD28- effector T cells and an increase of R3-specific CD8+ T cells. In the higher peptide dose cohort three patients showed positive clinical effects. However, higher doses of peptide do not improve the frequency and intensity of immune responses in this clinical trial and might induce immune tolerance.In this work, we investigated the co-existence of serological immune responses against RHAMM detected by a RHAMM-specific ELISA of patients with AML, MDS and multiple myeloma treated in these two peptide vaccination trials. We correlated these results to specific T cell responses of CD8-positive T cells measured by ELISpot assays for interferon gamma and Granzyme B, tetramer staining and chromium release assays. Moreover, these results were compared to the frequency of regulatory T cells. 4/19 patients have a positive serological immune response in ELISA assay, all of these patients developed also strong specific CD8-positive T cell responses during peptide vaccination detected by ELISpot assays and tetramer staining. As expected, peptide vaccination did not result in the induction of humoral immune responses. In further ELISA assays we measured IL-2 and IL-10 levels in the sera of the patients before and three weeks after four vaccinations. While IL-10 levels remained at a rather low level over the time of vaccination, we detected an increase of IL-2 up to the five-fold of the initial levels in four of ten patients.Moreover, we performed a proteome array to detect cytokine and chemokine regulation in sera of patients vaccinated in these two trials during and after RHAMM-R3 peptide vaccination. 36 cytokines, chemokines and acute phase proteins were measured and both cohorts vaccinated with different peptide doses were compared.Taken together, RHAMM-R3 peptide vaccination induced both immunological and clinical responses. Co-existence of immune responses of CD4-positive T cells against the target RHAMM seems to be important for an induction of strong immune responses of CD8-positive T cells. Disclosures:No relevant conflicts of interest to declare.