Local Humoral Immune Response Research Articles

Escherichia coli enterotoxigénica y enteroagregativa: prevalencia, patogénesis y modelos múridos.

Enterotoxigenic (ETEC) and enteroaggregative Escherichia coli (EAEC) pathotypes are important etiological agents causative of diarrhea in children younger than 5 years of age in Mexico and in developing countries, where they cause numerous deaths. Both have been associated with delayed growth in children and are the main causative agents of traveler's diarrhea. The pathogenesis of both bacteria starts by adhering to the intestinal epithelium by means of fimbriae, called colonization factors in human ETEC isolates and aggregative adherence fimbriae in EAEC isolates. Once ETEC adheres to the enterocyte, it produces one or both of its toxins and induces the secretion of chloride and sodium ions and water into the intestinal lumen, producing its characteristic watery diarrhea. EAEC binds to the intestinal epithelium forming a biofilm, induces the production of mucus, releases its toxins and promotes inflammation. EAEC and ETEC infection models with wild-type C57BL/6 and CD40 ligand-deficient mice (with intact microbiota), respectively, revealed that undernutrition and low-zinc diet increases EAEC infection, causing growth retardation, and that ETEC colonizes, persists and induces local and systemic humoral immune response.

Mouse intestinal microbiota reduction favors local intestinal immunity triggered by antigens displayed in Bacillus subtilis biofilm

BackgroundWe previously engineered Bacillus subtilis to express an antigen of interest fused to TasA in a biofilm. B. subtilis has several properties such as sporulation, biofilm formation and probiotic ability that were used for the oral application of recombinant spores harboring Echinococcus granulosus paramyosin and tropomyosin immunogenic peptides that resulted in the elicitation of a specific humoral immune response in a dog model.ResultsIn order to advance our understanding of the research in oral immunization practices using recombinant B. subtilis spores, we describe here an affordable animal model. In this study, we show clear evidence indicating that a niche is required for B. subtilis recombinant spores to colonize the densely populated mice intestinal microbiota. The reduction of intestinal microbiota with an antibiotic treatment resulted in a positive elicitation of local humoral immune response in BALB/c mice after oral application of recombinant B. subtilis spores harboring TasA fused to E. granulosus (102-207) EgTrp immunogenic peptide. Our results were supported by a lasting prevalence of spores in mice feces up to 50 days after immunization and by the presence of specific secretory IgA, isolated from feces, against E. granulosus tropomyosin.ConclusionsThe reduction of mouse intestinal microbiota allowed the elicitation of a local humoral immune response in mice after oral application with spores of B. subtilis harboring immunogenic peptides against E. granulosus.

Cytokines, Antibodies, and Histopathological Profiles during Giardia Infection and Variant-Specific Surface Protein-Based Vaccination.

Giardiasis is one of the most common human intestinal diseases worldwide. Several experimental animal models have been used to evaluate Giardia infections, with gerbils (Meriones unguiculatus) being the most valuable model due to their high susceptibility to Giardia infection, abundant shedding of cysts, and pathophysiological alterations and signs of disease similar to those observed in humans. Here, we report cytokine and antibody profiles both during the course of Giardia infection in gerbils and after immunization with a novel oral vaccine comprising a mixture of purified variant-specific surface proteins (VSPs). Transcript levels of representative cytokines of different immune profiles as well as macro- and microtissue alterations were assessed in Peyer's patches, mesenteric lymph nodes, and spleens. During infection, cytokine responses showed a biphasic profile: an early induction of Th1 (gamma interferon [IFN-γ], interleukin-1β [IL-1β], IL-6, and tumor necrosis factor [TNF]), Th17 (IL-17), and Th2 (IL-4) cytokines, together with intestinal alterations typical of inflammation, followed by a shift toward a predominant Th2 (IL-5) response, likely associated with a counterregulatory mechanism. Conversely, immunization with an oral vaccine comprising the entire repertoire of VSPs specifically showed high levels of IL-17, IL-6, IL-4, and IL-5, without obvious signs of inflammation. Both immunized and infected animals developed local (intestinal secretory IgA [S-IgA]) and systemic (serum IgG) humoral immune responses against VSPs; however, only infected animals showed evident signs of giardiasis. This is the first comprehensive report of cytokine expression and anti-Giardia antibody production during infection and VSP vaccination in gerbils, a reliable model of the human disease.

Targeted Delivery of Toxoplasma gondii Antigens to Dendritic Cells Promote Immunogenicity and Protective Efficiency against Toxoplasmosis.

Toxoplasmosis is a major public health problem and the development of a human vaccine is of high priority. Efficient vaccination against Toxoplasma gondii requires both a mucosal and systemic Th1 immune response. Moreover, dendritic cells play a critical role in orchestrating the innate immune functions and driving specific adaptive immunity to T. gondii. In this study, we explore an original vaccination strategy that combines administration via mucosal and systemic routes of fusion proteins able to target the major T. gondii surface antigen SAG1 to DCs using an antibody fragment single-chain fragment variable (scFv) directed against DEC205 endocytic receptor. Our results show that SAG1 targeting to DCs by scFv via intranasal and subcutaneous administration improved protection against chronic T. gondii infection. A marked reduction in brain parasite burden is observed when compared with the intranasal or the subcutaneous route alone. DC targeting improved both local and systemic humoral and cellular immune responses and potentiated more specifically the Th1 response profile by more efficient production of IFN-γ, interleukin-2, IgG2a, and nasal IgA. This study provides evidence of the potential of DC targeting for the development of new vaccines against a range of Apicomplexa parasites.

Mucosal Administration of Cycle-Di-Nucleotide-Adjuvanted Virosomes Efficiently Induces Protection against Influenza H5N1 in Mice.

The need for more effective influenza vaccines is highlighted by the emergence of novel influenza strains, which can lead to new pandemics. There is a growing population of susceptible subjects at risk for severe complications of influenza, such as the elderly who are only in part protected by current licensed seasonal vaccines. One strategy for improving seasonal and pandemic vaccines takes advantage of adjuvants to boost and modulate evoked immune responses. In this study, we examined the capacity of the recently described adjuvant cyclic di-adenosine monophosphate (c-di-AMP) to serve as an adjuvant for improved mucosal influenza vaccines, and induce effective protection against influenza H5N1. In detail, c-di-AMP promoted (i) effective local and systemic humoral immune responses, including protective hemagglutination inhibition titers, (ii) effective cellular responses, including multifunctional T cell activity, (iii) induction of long-lasting immunity, and (iv) protection against viral challenge. Furthermore, we demonstrated the dose-sparing capacity of the adjuvant as well as the ability to evoke cross-clade protective immune responses. Overall, our results suggest that c-di-AMP contributes to the generation of a protective cell-mediated immune response required for efficacious vaccination against influenza, which supports the further development of c-di-AMP as an adjuvant for seasonal and pandemic influenza mucosal vaccines.

Local and systemic humoral immune response in farmed Atlantic salmon (Salmo salar L.) under a natural amoebic gill disease outbreak

Amoebic gill disease (AGD), caused by the protozoan parasite Neoparamoeba perurans, is one of the most significant infectious diseases for Atlantic salmon (Salmo salar L.) mariculture. The present study investigated the humoral immune response (both local in gill mucus and systemic in serum) of farmed Atlantic salmon naturally infected with N. perurans in commercial sea pens, at two different stages of the disease and after freshwater treatment. Parameters analysed included activity of immune related enzymes (i.e. lysozyme, peroxidase, protease, anti-protease, esterase, alkaline phosphatase), IgM levels, and the terminal carbohydrate profile in the gill mucus. Overall, greater variations between groups were noted in the immune parameters determined in gill mucus than the equivalent in the serum. In gill mucus, IgM levels and peroxidase, lysozyme, esterase and protease activities were decreased in fish showing longer exposure time to the infection and higher disease severity, then showed a sequential increase after treatment. Results obtained highlight the capacity of gills to elicit a local response to the infection, indicate an impaired immune response at the later stages of the disease, and show partial reestablishment of the host immune status after freshwater treatment. In addition to providing data on the humoral response to AGD, this study increases knowledge on gill mucosal humoral immunity, since some of the parameters were analysed for the first time in gill mucus.

Immune response and distribution of antigen in chickens after infection LPAIV (H4N6)

ObjectiveTo study the immune response in chicken on the administration ofLPAIV isolated from the natural reservoir.IntroductionInfluenza is a serious problem for the health of people, animals andbirds. Therefore, comprehensive study of influenza virus, its naturalreservoir, pathogenesis and immune response will provide furtheropportunity to ensure protection for animals, birds and people fromthis infection.MethodsFour-week-old commercial chickens were intranasally inoculatedwith a H4N6 LPAIV A/Garganey/Chervonooskilske/4-11/2009(H4N6), isolated from the cloacal swab of clinically healthy garganeyin 2009 in Ukraine. Cecum, spleen, lung, and trachea samples werecollected from infected chickens on 1 - 14 dpi and examined byimmunohistochemical and virology techniques. On these days,we collected blood samples for serological analysis. Detection ofantibodies to avian influenza virus subtype H4 was performed withchicken serum samples by HI test and ELISA. The studies were doneaccording to IACUC.ResultsUpon intravenous and intranasal infection with this virus(A/Garganey/Chervonooskilske/4-11/2009), no clinical signs wereobserved in chickens and no pathological changes were found atnecropsy. Infection of poultry with this virus provoked an antibodyresponse at 10 days after intranasal inoculation which ranged from1:8 to 1:32 serum antibody titers. Only 2 of 5 chickens were positiveby the HI test and 3 of 5 were positive by ELISA at intranasalinoculation. All 10 chickens were positive both by HI test and byELISA after intravenous inoculation. Specific antibodies (HI test) toinfluenza virus H4 were detected in titer ranges of 1:128 to 1:1024.In immunohistological studies, the respiratory tract organs (lungsand trachea) showed higher level of humoral immunity (IgM, IgG,IgA-expressing cells) in the lung compared to the trachea. Also,indicators of cell mediated immunity as measured by the CD4 andmacrophage markers were higher in LPAIV-infected chickens inthe lungs at 14 days post infection compared to uninfected chickens.Lymphocytes expressing CD8 were increased starting 7dpi.The chickens in the infected group showed 2 times higher levels ofCD8 cells compared to the control chickens. IFN-γtranscripts wereobserved in the AI-infected chickens starting at 7dpi that coincideswith the increasing level ofCD4 cells. The number of lymphocytes which secrete IL-2 andIL-15 in AI-infected chickens were in general 1.5 to 2 times highercompared to the uninfected chickens. In AIV-infected chickens, thelevel of cells expressing IFN-γ, IL-2, and IL-15 increased at 7-daysafter infection. The peak time coincided with a period of increasingCD8 cells. However, there was no significant difference in thesecytokine levels between the AIV-infected and uninfected groups.In the cecum, lower levels of CD4 cells were seen on 5 dpi butlevels slowly increased from 7 dpi to 14 dpi following AIV infection.In the ceca, a significant increase in the number of cells expressingIgM and IgG was found.LPAIV infection induced an increase in macrophages andlymphocytes expressing CD4 and CD8 in the spleen throughout theperiod examined in this study indicating their role in host responseto viral infection. The levels of macrophages in chickens of AIV-infected group were 2 times higher than the control after 1 dpi.ConclusionsAlthough infection with a LPAIV did not cause obvious clinicaldisease, viral replication was detected in the trachea and spleen andboth local and systemic cellular and humoral immune responses wereelicited in these LPAIV-infected chickens. Our results indicate thepotential possibility for infection of poultry with viruses isolated fromwild birds. But currently it is not completely known why some virusesfrom wild birds can cause infection in poultry, while others can not.Further study of the immune response will enable us to determine thefeatures of the pathogenesis of low pathogenic avian influenza.

Association of Local Intrapulmonary Production of Antibodies Specific to Donor Major Histocompatibility Complex Class I With the Progression of Chronic Rejection of Lung Allografts.

Antibody-mediated rejection may lead to chronic lung allograft dysfunction, but antibody-mediated rejection may develop in the absence of detectable donor-specific antibody (DSA) in recipient serum. This study investigated whether humoral immune responses develop not only systemically but locally within rejected lung allografts, resulting in local production of DSA. Lewis rats received orthotopic left lung transplantation from Lewis (syngeneic control) or Brown-Norway (major histocompatibility complex-mismatched allogeneic) donor rats. Rats that underwent allogeneic lung transplantation were subsequently administered cyclosporine until day 14 (short immunosuppression) or day 35 (long immunosuppression). The lung grafts and spleens of recipient animals were tissue cultured for 4 days, and the titer of antibody against donor major histocompatibility complex molecules was assayed by flow cytometry. Explanted lung grafts were also evaluated pathologically. By day 98, DSA titers in supernatants of lung graft (P = 0.0074) and spleen (P = 0.0167) cultures, but not serum, from the short immunosuppression group were significantly higher than titers in syngeneic controls. Cultures and sera from the long immunosuppression group showed no production of DSA. Microscopically, the lung grafts from the short immunosuppression group showed severe bronchiole obliteration and parenchymal fibrosis, along with lymphoid aggregates containing T and B cells, accompanying plasma cells. These findings suggestive of local humoral immune response were not observed by days 28 and 63. DSA can be locally produced in chronically rejected lung allografts, along with intragraft immunocompetent cells. Clinical testing of DSA in serum samples alone may underestimate lung allograft dysfunction.

Protective immune responses during prepatency in goat kids experimentally infected with Eimeria ninakohlyakimovae

During the first schizogony, the goat coccidia Eimeria ninakohlyakimovae develops macroschizonts in lacteal duct endothelial cells, whose rupture leads to severe ileal damage and clinical signs during the prepatent period. The immune response elicited against early stages of the parasite development still requires to be investigated. In the present study we have evaluated immune reactions in goat kids primary- and challenged-infected with Eimeria ninakohlyakimovae, and sacrificed during prepatency (7days after challenge). The oocyst output during the primary infection, body weight and clinical condition of all the animals were examined and, at the end of the experiment, all the goat kids were euthanized and subjected to necropsy. Samples were taken from different sections of the ileum, colon and mesenteric lymph nodes (MLN) of primary- and challenged E. ninakohlyakimovae-infected animals. Intestinal leukocyte subpopulations were characterized in E. ninakohlyakimovae-infected mucosa and counts of lymphocytes, eosinophils, polymorphonuclear neutrophils (PMN), globular leukocytes and mast cells were recorded. Additionally, gene expression of caprine IL-2, IL-4, IL-10 and INFγ of ileal, colonic and MLN tissues were performed, as well as the immunohistochemical characterization of immune cells. The E. ninakohlyakimovae primary infection resulted in moderate to severe enteritis with different degrees of diarrhoea and was accompanied by high OPG counts and an increase of most immune cells analyzed when compared to uninfected control animals. Furthermore, eosinophil-, lymphocyte-, globular leukocyte- and mast cell-counts were significantly higher in the challenge group compared to the primary infected animals, whilst the opposite was true for PMN counts. The challenge infection was also associated with moderate increased levels of local mucosal IgA. Interestingly, the number of immature schizonts found at the ileal mucosa was statistically higher in the challenge infected group compared to the challenged control animals. Furthermore, in the challenged E. ninakohlyakimovae-infected animals a significantly higher number of mucosal CD4+ and CD8+ lymphocytes were observed, indicating that these T cell subpopulations might be involved in protective host immune response elicited against early stages of parasite development. The immune response was however very complex, as antigen presenting cells and other effector cell populations of the innate immune system, as well as certain cytokines, were involved. In summary, the results of this study contribute to the better understanding of local cellular and humoral immune responses against caprine E. ninakohlyakimovae, particularly during the prepatency.

Potential use of local and systemic humoral immune response parameters to forecast Mycoplasma hyopneumoniae associated lung lesions.

Immunopathological events are key for the development of enzootic pneumonia (EP), which is macroscopically observed as cranioventral pulmonary consolidation (CVPC). This study aimed to investigate the putative association between the humoral immune response against Mycoplasma hyopneumoniae (M. hyopneumoniae) and prevalence and extension of CVPC in 1) experimentally infected pigs, 2) slaughtered pigs and 3) sequentially necropsied pigs in a longitudinal study. CVPC was scored by means of the European Pharmacopoeia recommended methodology. Specific IgG, IgG1 and IgG2 antibodies were assessed in serum. In addition, mucosal IgG and IgA antibodies were analyzed in broncho-alveolar lavage fluid (BALF) from experimentally challenged pigs. The systemic humoral immune response in experimentally infected pigs was delayed in onset whereas humoral respiratory mucosal immune response appeared more rapidly but declined earlier. Although low, BALF IgG antibodies showed the highest correlation with CVPC scores (r = 0.49, p<0.05). In slaughter-aged pigs, both percentage of lungs with CVPC and mean lung lesion score were significantly higher in M. hyopneumoniae seropositive farms compared to the seronegative ones (p<0.001). Similarly, seropositive sequentially necropsied pigs showed more severe CVPC than seronegative ones. Overall, mean serological values might help to forecast prevalence and severity of EP-like lung lesions using a population based approach. Remarkably, the specific systemic humoral immune response was found to be predominated by the IgG2 subclass, suggesting a dominant Th1-mediated immune response to M. hyopneumoniae.

Induction of Both Local Immune Response in Mice and Protection in a Rabbit Model by Intranasal Immunization with Modified Vaccinia Ankara Virus Expressing a Secreted Form of Bovine Herpesvirus 1 Glycoprotein D.

In this study, we evaluated the immunogenicity and efficacy of mucosal delivery of a recombinant modified vaccinia Ankara virus (MVA) expressing the secreted version of bovine herpesvirus type 1 (BoHV-1) glycoprotein D (MVA-gDs) without addition of adjuvant in two animal models. First, we demonstrated the capability of MVA-gDs of inducing both local and systemic anti-gD humoral immune response after intranasal immunization of mice. Then, we confirmed that two doses of MVA-gDs administered intranasally to rabbits induced systemic anti-gD antibodies and conferred protection against BoHV-1 challenge. Our results show the potential of using MVA as a vector for the rational design of veterinary vaccines capable of inducing specific and protective immune responses both at local and systemic level.

Directed chromosomal integration and expression of porcine rotavirus outer capsid protein VP4 in Lactobacillus casei ATCC393.

Using two-step plasmid integration in the presence of 5-fluorouracil (5-FU), we developed a stable and markerless Lactobacillus casei strain for vaccine antigen expression. The upp of L. casei, which encodes uracil phosphoribosyltransferase (UPRTase), was used as a counterselection marker. We employed the Δupp isogenic mutant, which is resistant to 5-FU, as host and a temperature-sensitive suicide plasmid bearing upp expression cassette as counterselectable integration vector. Extrachromosomal expression of UPRTase complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. The resultant genotype can either be wild type or recombinant. The efficacy of the system was demonstrated by insertion and expression of porcine rotavirus (PRV) VP4. To improve VP4 expression, we analyzed L. casei transcriptional profiles and selected the constitutive highly expressed enolase gene (eno). The VP4 inserted after the eno termination codon were screened in the presence of 5-FU. Using genomic PCR amplification, we confirmed that VP4 was successfully integrated and stably inherited for at least 50 generations. Western blot demonstrated that VP4 was steadily expressed in medium with different carbohydrates. RT-qPCR and ELISA analysis showed that VP4 expression from the chromosomal location was similar to that achieved by a plasmid expression system. Applying the recombinant strain to immunize BALB/c mice via oral administration revealed that the VP4-expressing L. casei could induce both specific local and systemic humoral immune responses in mice. Overall, the improved gene replacement system represents an efficient method for chromosome recombination in L. casei and provides a safe tool for vaccine production.

Abstract A124: Tertiary lymphoid structures: Predictors of effective antitumor immunity in human breast cancer?

Abstract The clinical relevance of tumor infiltrating lymphocytes (TIL) in breast cancer (BC) is now broadly accepted; however, the relationship between protective immunity, observed in some patients, and critical features of lymphoid subset composition and organization are largely unknown. Our recent work revealed that tertiary lymphoid structures (TLS), principally composed of T and B cells, present in the peri-tumoral regions of BC are associated with increased TIL infiltration in the tumor bed and good clinical outcomes in both the neo-adjuvant and adjuvant settings. To gain insight into the immune components linked with a significant TIL and TLS presence, we initiated a prospective study of T and B cell TIL. Breast tissues from tumors (current n=483) and matched non-adjacent non-tumor tissues (NANT) as well as normal tissue from mammary reductions (n=49) were systematically immunophenotyped by flow cytometry on the day of surgery. Primary tumor supernatants derived from the tissue homogenates (non-enzymatic) and plasma from all patients were stored for cytokine/chemokine and immunoglobulin analysis. TIL organization and spatial distribution was analyzed on paraffin sections using immunohistochemistry (IHC) and immunofluorescence (IF). The fresh tissue analyses revealed that TIL distribution in BC forms a continuum. The infiltration levels detected in normal tissue and NANT were used to define cutoffs that discriminate between TIL positive (TILpos) and negative (TILneg) tumors. TILneg tumors are defined as those with a TIL density &amp;lt;99th percentile of the lymphocyte density in normal breast tissue. TILpos tumors include TIL-high (TILhi) where TIL density is &amp;gt;99th percentile of lymphocyte density in NANT and TIL-intermediate (TILint). Applying these thresholds to the TIL infiltration levels identified approximately 25% of tumors as TILneg with infiltrating lymphocytes similar to normal breast tissue. The TILpos tumors (75%) are subdivided into 36% TILint and 39% TILhi tumors. TILpos tumors are characterized by an increase in CD4+ T cells and CD19+/CD20+ B cells. The median CD4/CD8 ratio was &amp;gt;1 in TILpos compared to &amp;lt;1 in TILneg tumors and NANT. CD4+ and CD8+ T cells were predominantly CD45RO+ memory cells with a significant proportion expressing PD-1. All stages of B cell differentiation were detected in the infiltrate; however, memory B cells were significantly increased in BC (50%) compared to normal tissues (&amp;lt;15%) with the highest levels seen in TILhi tumors. An increase in centroblasts and centrocytes, the germinal center (GC) B cells, was associated with Tfh cells, both resident in peri-tumoral TLS. Their presence was positively correlated with antibody-secreting cells, suggesting local humoral immune responses are generated in TLS, which is supported by the increased immunoglobulin levels detected in tumor supernatants compared to NANT/normal tissue supernatants. Closer examination of Tfh cells infiltrating BC indicates that CXCL13-producing Tfh TIL vary from Tfh cells in secondary lymphoid organs such as tonsils. Tfh TIL express the highest levels of some GC Tfh cell markers (PD-1, CD200 and TIGIT) but intermediate levels of others (ICOS and BCL6) and are essentially negative for CXCR5. Closer examination of Tfh cells in the tumor bed revealed that they directly contact CD20+ B cells, IgG+ and IgA+ plasma cells as well as CXCL13–CD8+ TIL. Taken together, our data suggest that active cross-talk between adaptive immune cells in the tumor microenvironment may be key to establishing immunological memory sufficiently robust to control residual BC over the long term. These data further signify that TLS and/or the presence of GC-associated Tfh and B cells may be important biomarkers that identify effective anti-tumor immunity, which can ultimately be used to independently grade therapeutic responses. Citation Format: Soizic Garaud, Laurence Buisseret, Chunyan Gu-Trantien, Gert Van den Eynden, Alexandre de Wind, Edoardo Migliori, Roberto Salgado, Denis Larsimont, Martine Piccart, Karen Willard-Gallo. Tertiary lymphoid structures: Predictors of effective antitumor immunity in human breast cancer? [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A124.

Gastric cancer progression associated with local humoral immune responses.

BackgroundAlthough the association between H. pylori and gastric cancer has been well described, the alterations studies are scarce in the humoral immune response in specific anatomical areas of stomach and during the stages of gastric cancer. The aim in this study was to determine the influence of humoral immune responses against H. pylori infection on gastric carcinoma.MethodsWe selected 16 gastric cancer cases and approximately one matched control per case at the National Institute of Medical Sciences and Nutrition Salvador Zubirán (INCMNSZ); all the cases met the inclusion criteria for the study. We obtained three biopsies from each patient and from each of the predetermined regions of the stomach: antrum, angular portion, corpus, and fundus. From the patients with gastric cancer, additional biopsy specimens were obtained from tumor mid-lesion and tumor margin, and additional specimens were collected at least 2 and 5 cm from the tumor margin. We compared IgA levels against H. pylori in each area of stomach between cases and controls as well as between early and advanced stages of gastric cancer.ResultsIgA values were strikingly elevated in cancer cases compared with control subjects; a value that was even higher in the distant periphery of tumor but was remarkably decreased toward the carcinoma lesion. The advanced stages of gastric cancer demonstrated the relapse of the humoral immune response in the mid-lesion region of the tumor compared with the tumor margins and adjacent non-tumor tissue.ConclusionsGastric cancer is characterized by progressive accumulation of a concentrated, specific IgA response against H. pylori, beginning with an abnormal increase in the entire stomach but particularly in the adjacent non-tumor tissue. Thus, it is possible that this strong immune response also participates in some degree in the damage and in the development of gastric cancer to some extent.

Immunogenic properties of the surface layer precursor of Clostridium difficile and vaccination assays in animal models

Clostridium difficile is an opportunistic pathogen causing gut inflammation generally associated with an intestinal dysbiosis due to antibiotics. Several virulence factors have been identified as playing a key role in gut colonization. The surface-layer proteins, comprised of two proteins, the high molecular weight SlpA (HMW-SLP) and the low molecular weight SlpA (LMW-SLP), are the most abundant proteins on the C. difficile surface. These two proteins are derived from the Cwp84-mediated cleavage of a single precursor protein SlpA. In this study, we assessed the immunogenic properties of a recombinant SlpA precursor derived from a toxigenic C. difficile strain (630) and its protective effect as a vaccine antigen co-administered with the cholera toxin as an adjuvant in both hamster and mouse models.First, we confirmed the immunogenicity of SlpA in humans. Sera from patients with C. difficile infection were analyzed by ELISA. Patients with CDI have a greater number of SlpA antibodies than healthy patients, confirming the immunogenicity of this protein during the pathogenic process. Then, rectal vaccination assays were performed in both conventional hamsters and mice. The animals’ sera were sampled before and after vaccination, and were analyzed by ELISA. In addition, in the mouse model, feces were sampled after vaccination and IgA directed against SlpA were detected by ELISA. In both models, the intestinal colonization was evaluated by fecal bacterial count after challenge. Intra-rectal vaccination with SlpA and cholera toxin as an adjuvant induced a local and systemic humoral immune response in mice and hamsters potentially responsible for the weak decrease of C. difficile colonization in mice and the partial protection observed in a lethal-hamster model.

Abstract 1287: Characterization of B cells infiltrating human breast cancer

Abstract Recent advances in tumor immunology have shown that an important link exists between tumor infiltrating lymphocytes (TIL) and patient outcome. In human breast cancer (BC), more extensive lymphocyte infiltration is associated with a better prognosis and also predicts responses to pre-operative chemotherapy. TIL are principally composed of T cells; however, approximately 20% of BC patients have a significant B cell infiltration, which can be diffuse and/or aggregated in stroma-located tertiary lymphoid structures (TLS). B cell infiltration in human BC has been associated with better survival and a longer disease-free interval, although the pro-/anti-tumor activities of B cells remain under debate. We undertook this study to more fully characterize the B cell infiltrate in human BC and the role humoral immunity potentially plays in generating effective anti-tumor immune responses. Flow cytometric analysis of fresh breast tissue homogenates detected an increased density of infiltrating B cells in all untreated primary BC (n = 223) compared with normal (mammary reductions; n = 53) and non-tumor non-adjacent (NANT; n = 198) breast tissues. This increase is not observed in BC tissue from metastatic and recurrent BC nor neoadjuvant-treated primary tumors. Potential correlations between infiltrating B cell density and clinicopathological parameters were examined, revealing that B cell density was associated with Ki67, ER and PgR status but not with age, tumor size, nodal status, grade, vascular invasion, HER-2 status, stage, histotype, subtype or in situ lesions. B cell infiltration was also correlated with the number of infiltrating CD4+ and CD8+ T cells and CD14+ monocytes. All stages of B cell differentiation were detected in the infiltrate; however, memory B cells significantly increased in BC compared to normal breast tissues. An increase in centroblasts and centrocytes, the germinal center B cells, was associated with a TFH cell presence (our data previously demonstrating that TFH cells predict good clinical outcomes). Furthermore, the presence of centroblasts-centrocytes and TFH cells was positively correlated with antibody-secreting cells, suggesting local humoral immune responses are generated in TLS. This hypothesis is supported by the increased immunoglobulin secretion detected in primary supernatants from tumors compared with normal breast tissues. Embedded BC tissues, examined by confocal microscopy, confirm that infiltrating B cells are principally located in TLS, which are composed of marginal, follicular mantle and germinal center zones surrounded by T cells. These data show that B cell infiltration is correlated with the overall extent of TIL and their presence likely functions to generate a humoral immune response adjacent to the tumor bed. B cells in extensively infiltrated tumors potentially play other important functional roles in generating effective anti-tumor immune responses, an aspect currently under investigation. Citation Format: Soizic Garaud, Laurence Buisseret, Céline Naveaux, Anaïs Boisson, Jean-Nicolas Lodewyckx, Hugues Duvillier, Ligia Craciun, Denis Larsimont, Karen Willard-Gallo. Characterization of B cells infiltrating human breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1287. doi:10.1158/1538-7445.AM2015-1287

St. Croix sheep produce a rapid and greater cellular immune response contributing to reduced establishment of Haemonchus contortus

The objective of this study was to determine breed differences in immune response shortly following Haemonchus contortus infection. Peripheral and local cellular and humoral immune responses were evaluated in 24 St. Croix hair lambs and 24 Dorset×(Finn–Rambouillet) wool lambs at 0, 3, 5 and 7 days after infection with 10,000 L3 H. contortus larvae. Blood samples taken immediately before harvest revealed no differences in circulating effector cell populations, yet there were significant differences in levels of circulating neutrophils. Across all time points, hair lambs had a higher average circulating neutrophil concentration (3018cells/μl) than wool lambs (1818cells/μl; P<0.05). Infected hair lambs also had greater serum total-IgA compared to wool lambs (1.8 vs 0.9mg/ml; P=0.006). Breeds did not differ in eosinophil or globule leukocyte (GL) counts in abomasal tissue, but infiltration of these cell populations increased with time. Globule leukocyte counts peaked at day 3 after infection whereas eosinophil numbers continued to increase to day 7 after infection. When averaged across all time points, abomasal neutrophil counts were higher in hair lambs (831cells/mm2), than wool lambs (561cells/mm2; P<0.0001). Total abomasal lymph node (ALN) weight increased exponentially from 2.60g at day 0 to 6.57g by day 7 in hair lambs whereas ALN weight only marginally increased in wool lambs and was significantly lower than hair lambs by day 7 (P=0.0003). This result suggests a greater expansion of lymphocytes in the ALN promoting early development of antigen-specific acquired immune responses in hair lambs. Greater IgA production and infiltration of immune cells to the abomasal mucosa at an earlier stage of infection may limit establishment of adult parasites and thereby shorten the duration and severity of infection.

Trichomonas vaginalis infection induces vaginal CD4+ T-cell infiltration in a mouse model: a vaccine strategy to reduce vaginal infection and HIV transmission.

Complications related to the diagnosis and treatment of Trichomonas vaginalis infection, as well as the association between T. vaginalis infection and increased transmission of and susceptibility to human immunodeficiency virus, highlight the need for alternative interventions. We tested a human-safe, aluminum hydroxide-adjuvanted whole-cell T. vaginalis vaccine for efficacy in a BALB/c mouse model of vaginal infection. A whole-cell T. vaginalis vaccine was administered subcutaneously to BALB/c mice, using a prime-boost vaccination schedule. CD4(+) T-cell infiltration in the murine vaginal tissue and local and systemic levels of immunoglobulins were measured at time points up to 4 weeks following infection. Vaccination reduced the incidence and increased the clearance of T. vaginalis infection and induced both systemic and local humoral immune responses. CD4(+) T cells were detected in vaginal tissues following intravaginal infection with T. vaginalis but were not seen in uninfected mice. The presence of CD4(+) T cells following T. vaginalis infection can potentially increase susceptibility to and transmission of human immunodeficiency virus. The vaccine induces local and systemic immune responses and confers significantly greater protection against vaginal infection than seen in unvaccinated mice (P < .005). These data support the potential for a human vaccine against T. vaginalis infection that could also influence the incidence of human immunodeficiency virus infection.

Comparison of immune responses to intranasal and intrapulmonary vaccinations with the attenuated Mycoplasma hyopneumoniae 168 strain in pigs.

The aim of this study was to evaluate the immune responses to intranasal and intrapulmonary vaccinations with the attenuated Mycoplasma hyopneumoniae (Mhp) 168 strain in the local respiratory tract in pigs. Twenty-four pigs were randomly divided into 4 groups: an intranasal immunization group, an intrapulmonary immunization group, an intramuscular immunization group and a control group. The levels of local respiratory tract cellular and humoral immune responses were investigated. The levels of interleukin (IL)-6 in the early stage of immunization (P<0.01), local specific secretory IgA (sIgA) in nasal swab samples (P<0.01); and IgA- and IgG-secreting cells in the nasal mucosa and trachea were higher after intranasal vaccination (P<0.01) than in the control group. Interestingly, intrapulmonary immunization induced much stronger immune responses than intranasal immunization. Intrapulmonary immunization also significantly increased the secretion of IL-6 and local specific sIgA and the numbers of IgA- and IgG-secreting cells. The levels of IL-10 and interferon-γ in the nasal swab samples and the numbers of CD4+ and CD8+ T lymphocytes in the lung and hilar lymph nodes were significantly increased by intrapulmonary immunization compared with those in the control group (P<0.01). These data suggest that intrapulmonary immunization with attenuated Mhp is effective in evoking local cellular and humoral immune responses in the respiratory tract. Intrapulmonary immunization with Mhp may be a promising route for defense against Mhp in pigs.

Development of vaccine-induced immunity against TRT in turkeys depends remarkably on the level of maternal antibodies and the age of birds on the day of vaccination.

BackgroundAvian Metapneumovirus (aMPV) infections are a huge economical issue for the poultry industry worldwide. Although maternal antibodies do not protect turkey poults against turkey rhinotracheitis (TRT), almost no studies have been conducted so far regarding the impact of these antibodies on vaccine induced immunity development against aMPV infection. We conducted four experiments on commercial turkeys aimed at comparing local humoral and cell mediated immune response of maternally delivered anti-aMPV antibody positive (MDA+; Experiment I and II) and negative (MDA-; Experiment III and IV) turkeys following vaccination with an attenuated live aMPV subtype A vaccine at the day of hatch (Experiment I and III) or at two weeks of age (Experiment II and IV).ResultsRegardless of the birds’ age, vaccination of MDA- turkeys resulted in strong stimulation of CD8+ T lymphocytes in the Harderian gland and tracheal mucosa, whereas vaccination of MDA+ birds stimulated mainly CD4+ T cells in those structures. An increase in the level of anti-aMPV IgY antibodies was noted in the serum (but not in tracheal washings) as early as 7 days after vaccination, but only in birds possessing low levels (MDA+ birds vaccinated at 2 weeks of age) or no maternal anti-aMPV antibodies at the time of vaccination. In MDA+ turkeys vaccinated at hatch, the decrease in serum levels of maternal anti-aMPV antibodies proceeded faster (in comparison to control group), which, together with faster viral clearance, indicates that maternal antibodies can inhibit vaccine virus replication and influence the development of vaccine-induced immunity.ConclusionThis study provides the first documented evidence that the frequency of TRT outbreaks in the field and/or failure of TRT vaccination could be correlated with differences in the immunological status and/or age of vaccinated turkeys.