Secondary Antibody Response Research Articles

Effects of 2 size classes of intratracheally administered airborne dust particles on primary and secondary specific antibody responses and body weight gain of broilers: A pilot study on the effects of naturally occurring dust

We studied the effects of a concurrent challenge on slow-growing broilers with 1) airborne particles of 2 sizes: fine dust (smaller than 2.5 microns) and coarse dust (between 2.5 and 10 microns) that were directly collected from a broiler house and 2) lipopolysaccharide on intratracheal immunizations with the specific antigen human serum albumin (HuSA) and measured primary and secondary systemic (total) antibody responses and (isotype-specific) IgM, IgG, and IgA responses at 3 and 7 wk of age. All treatments affected immune responses at several ages, heart morphology, and BW gain, albeit the latter only temporarily. Dust particles significantly decreased primary antibody (IgT and IgG) responses to HuSA at 3 wk of age but enhanced IgM responses to HuSA at 7 wk of age. Dust particles decreased secondary antibody responses to HuSA, albeit not significantly. All of the birds that were challenged with dust particles showed decreased BW gain after the primary but not after the secondary challenge. Relative heart weight was significantly decreased in birds challenged with coarse dust, fine dust, lipopolysaccharide, and HuSA at 3 wk of age, but not in birds challenged at 7 wk of age. Morphology (weight, width, and length) of hearts were also affected by the dust challenge at 3 wk of age. The present results indicate that airborne dust particles obtained from a broiler house when intratracheally administered at an early age affect specific humoral immune responsiveness and BW gain of broilers to simultaneously administered antigens differently than when administered at a later age. The hygienic status of broiler houses at a young age may be of importance for growth and immune responsiveness, and consequently, for vaccine efficacy and disease resistance in broilers. The consequences of our findings are discussed.

PEGylated cationic liposomes robustly augment vaccine-induced immune responses: Role of lymphatic trafficking and biodistribution

Lymph nodes (LNs) are peripheral lymphoid organs essential for vaccine-induced immune responses. Although cationic liposomes have been documented as a novel adjuvant and vaccine delivery system, whether enhancing LN targeting would improve the efficiency of cationic liposome-formulated vaccines has not been elucidated yet. In the present study we investigated the effect of PEGylation on LN targeting and the immunogenicity of cationic liposome-formulated vaccines. DOTAP cationic liposomes were incorporated with 1 or 5mol% of DSPE-PEG2000 and labeled with near infrared fluorescent dyes. The lymphatic trafficking and biodistribution of different liposomes after subcutaneous (s.c.) injection were recorded using an in-vivo imaging system. The results showed that incorporation of 1mol% DSPE-PEG2000 not only accelerated the drainage of DOTAP liposomes into draining LNs, but also prolonged their LN retention and enhanced liposome uptake by resident antigen-presenting cells. On the other hand, although incorporating 5mol% of DSPE-PEG2000 into DOTAP liposomes enhanced their LN retention and uptake to a lesser extent, it prolonged blood circulation of DOTAP liposomes and increased their splenic accumulation. In addition, PEGylated DOTAP liposomes augmented primary and secondary anti-OVA antibody responses more potently than nonPEGylated DOTAP liposomes did. Hence, incorporating a small amount of DSPE-PEG2000 into DOTAP liposomes not only increased the passive LN targeting of DOTAP-formulated vaccines but also modulated their biodistribution in vivo, which consequently improved the efficiency of cationic liposome-formulated vaccines.

Soybean agglutinin coated PLA particles entrapping candidate vaccines induces enhanced primary and sustained secondary antibody response from single point immunization

Polylactide (PLA) polymer particles entrapping diphtheria toxoid (DT) or tetanus toxoid (TT) were formulated with surface coatings of soybean agglutinin to have dendritic cells (DCs) targeting ability through c-type lectin receptors (CLR). It was observed that soybean agglutinin coating resulted in more association of polymer particles with DCs. Immunization with soybean agglutinin coated polymer particles entrapping DT or TT elicited antibody response better than the plain particles entrapping antigens. Both for TT and DT, single point immunization of soybean agglutinin coated polymer particles along with alum resulted in very high antibody titers; much higher than that observed while immunizing with alum adsorbed antigens or admixture of particle entrapped antigens and alum. More interestingly, single point immunization with soybean agglutinin coated polymer particles also elicited very high secondary antibody response which sustained for more than six weeks in mice. Interactions of different polymeric microparticles formulations with DCs correlated with antibody response. Improved primary and sustained secondary antibody response from single point immunization of antigen entrapped soybean agglutinin coated particles was attributed to the N-linked glycan mediated targeting of polymer particles to DCs.

Effects of Different Levels of Organic and Inorganic Chromium on Growth Performance and Immunocompetence of Broilers under Heat Stress

This experiment was carried out to investigate the effects of different levels of organic and inorganic chromium on the performance, immune function and some serum mineral concentrations of broilers under heat stress condition (23.9-37°C cycling). A total of 150 one-day-old broiler chicks according to a completely randomized design were assigned into five treatment groups. Each treatment consisted of three replicates and each replicate contained ten chicks. Chicks were fed on corn-soybean meal basal diets with added different concentrations of chromium (0, 600 and 1,200 μg kg(-1) chromium chloride or 600 and 1,200 μg/kg chromium L-methionine) from 1 to 49 days of age. Humoral immunity was assessed by intravenous injection of 7% sheep red blood cell (SRBC) followed by evaluation of serum for antibody titers in primary and secondary responses. Cell-mediated immunity was assessed by the cutaneous basophil hypersensitivity (CBH) test to phytohemagglutinin (PHA)-P at day 32 and PHA-M at day 48. Heterophil/lymphocyte (H/L) ratio was also measured as a reliable indicator of stress. The body mass, feed intake and conversion ratio were not influenced by dietary chromium (P > 0.05). Dietary supplementation of both organic and inorganic chromium significantly increased primary and secondary antibody responses (P < 0.01), and also improved H/L ratio (P < 0.05), CBH response (P < 0.01) as well as relative weights of thymus (P < 0.05) and spleen (P < 0.01). Both dietary organic and inorganic chromium caused an increase in serum concentrations of Cr and Zn (P < 0.01), but decreased the serum concentration of Cu (P < 0.01). These results suggest that supplemental chromium especially in organic form offers a good management practice to reduce heat stress-related depression in immunocompetence of broiler chicks.

Vaccine induced antibodies to the first variable loop of human immunodeficiency virus type 1 gp120, mediate antibody-dependent virus inhibition in macaques

The role of antibodies directed against the hyper variable envelope region V1 of human immunodeficiency virus type 1 (HIV-1), has not been thoroughly studied. We show that a vaccine able to elicit strain-specific non-neutralizing antibodies to this region of gp120 is associated with control of highly pathogenic chimeric SHIV(89.6P) replication in rhesus macaques. The vaccinated animal that had the highest titers of antibodies to the amino terminus portion of V1, prior to challenge, had secondary antibody responses that mediated cell killing by antibody-dependent cellular cytotoxicity (ADCC), as early as 2 weeks after infection and inhibited viral replication by antibody-dependent cell-mediated virus inhibition (ADCVI), by 4 weeks after infection. There was a significant inverse correlation between virus level and binding antibody titers to the envelope protein, (R=-0.83, p=0.015), and ADCVI (R=-0.84 p=0.044). Genotyping of plasma virus demonstrated in vivo selection of three SHIV(89.6P) variants with changes in potential N-linked glycosylation sites in V1. We found a significant inverse correlation between virus levels and titers of antibodies that mediated ADCVI against all the identified V1 virus variants. A significant inverse correlation was also found between neutralizing antibody titers to SHIV(89.6) and virus levels (R=-0.72 p=0.0050). However, passive inoculation of purified immunoglobulin from animal M316, the macaque that best controlled virus, to a naïve macaque, resulted in a low serum neutralizing antibodies and low ADCVI activity that failed to protect from SHIV(89.6P) challenge. Collectively, while our data suggest that anti-envelope antibodies with neutralizing and non-neutralizing Fc(R-dependent activities may be important in the control of SHIV replication, they also demonstrate that low levels of these antibodies alone are not sufficient to protect from infection.

Heterozygous signal transducer and activator of transcription 3 mutations in hyper-IgE syndrome result in altered B-cell maturation

Heterozygous signal transducer and activator of transcription 3 mutations in hyper-IgE syndrome result in altered B-cell maturation

Different IgG-subclass distributions after whole-cell and acellular pertussis infant primary vaccinations in healthy and pertussis infected children

The distribution of IgG-subclasses provides insight in the immunological mechanisms of protection against whooping cough. We investigated the effect of Dutch whole-cell pertussis and acellular pertussis vaccines administered in infancy on the IgG-subclass distributions in healthy children aged 12 months, 4 years and 9 years as well as in children who have been infected with Bordetella pertussis. A fluorescent bead-based multiplex immunoassay was used for the measurement of IgG1, IgG2, IgG3 and IgG4 responses against pertussis toxin, filamentous heamagglutinin and pertactin. Although IgG1 was the predominant subclass for all pertussis antigens in both healthy and infected children, elevated IgG4 levels were only present in children who had received repeated number of acellular pertussis vaccinations. IgG2 and IgG3 antibodies did not contribute to the IgG response. No differences in IgG-subclasses between healthy vaccinated or infected children were found. The pertussis vaccine used for priming seems to determine the IgG-subclass composition elicited after a secondary antibody response either induced by pertussis vaccination or infection. The pronounced anti-pertussis IgG4 response might reflect the Th2-skewing of the immune response after aP vaccination.

Effect of methanolic extract of Mucuna pruriens seed on the immune response of mice

The effect of the methanolic extract of Mucuna pruriens seed was evaluated in mice with regards to delayed hypersensitivity reaction (DTR), primary and secondary antibody response and in vivo inflammatory leucocyte mobilization. The extract at 250 and 500 mg/kg produced significant (p < 0.05) inhibition of DTR in mice by 33.33% and 28.89%, respectively. The extract caused elevation of secondary SRBCs-specific antibody titre with antibody response being significantly (p < 0.05) increased at 250 and 500 mg/kg when compared with control. The extract at 250 and 500 mg/kg increased leucocyte mobilization significantly (p < 0.05) when compared with the control and levamisole-treated groups. At 100 mg/kg, the mobilization did not differ with the levamisole-treated group but was significantly (p < 0.05) higher than the control. The leucocyte mobilization was more of neutrophils than lymphocytes. The ability of the extract to inhibit the DTR, increase secondary antibody response and in vivo leucocyte mobilization was an indication that the extract probably influenced immune response in mice.

IL-21R is critical for the development of memory responses (150.9)

Abstract The development of long-term humoral immunity, characterized by the formation of high affinity memory B cells and long-lived plasma cells in the bone marrow, is a major goal of vaccination, but the mechanisms involved in the generation of long-term humoral immunity are not well understood. IL-21 can induce B cell differentiation into plasma cells and influence isotype switching to some subclasses of IgG. Using IL-21R KO mice, we show for the first time a role for the IL-21 pathway in the development of long-term humoral immunity. Antigen specific IgG serum responses to the T cell-dependent antigen, NP-CGG, were delayed in IL-21R KOs, but reached comparable titers to those measured in WT within 3 weeks. The formation of germinal centers, which give rise to long-lived plasma cells and memory B cells, was also delayed in IL-21R KO, but reached similar magnitudes as in WT within 2 weeks. IL-21R KO generated normal numbers of NP-specific IgG plasma cells in their bone marrow at ~1.5 months after immunization. NP-specific serum IgG antibody responses were of similar relative affinity in IL-21R KO and WT at this time, indicating that NP-specific B cells affinity matured in IL-21R KO. However, IL-21R KO failed to generate NP-specific secondary IgG antibody responses following rechallenge with NP-CGG. These results indicate that the IL-21 pathway is necessary for the formation of memory B cell responses but not high affinity long-lived plasma cells.

In vitro production of fully human monoclonal antibodies targeting therapeutic self-antigens (65.10)

Abstract Despite their role in autoimmune disease, not all autoreactive antibodies are harmful. Autoantibodies targeting tumor antigens, growth factors and their receptors are currently used therapeutically in cancer and autoimmunity. Ordinarily, healthy subjects do not exhibit strong secondary antibody responses to self-proteins, because most autoreactive B cells do not receive sufficient T cell help required for the germinal center reaction and for differentiation into IgG secreting cells. Our data demonstrate that rare, potentially therapeutic autoreactive B cells can be efficiently rescued from tonsil with EBV immortalization, and manipulated in vitro into secreting IgG, by supplying factors that mimic antigen stimulation and T cell help. Because this process is performed wholly in vitro, it constitutes a rapid and cost effective technology, resulting in the isolation of fully human antibodies. In addition, antibodies can be generated against highly toxic antigens, since immunization is unnecessary. We have developed two panels of human monoclonal antibodies targeting distinct self-antigens for use in cancer immunotherapy, one targeting the tumor antigen nucleolin and the other angiogenic placenta growth factor. We hypothesize that infection of B cells with the ubiquitous herpesvirus EBV might contribute to the persistance of autoreactive memory B cells in vivo, altering the B cell repertoire and resulting in the secretion of destructive and possibly of protective autoantibodies.

Immunotoxicity and disease resistance in Japanese quail (Corturnix coturnix japonica) exposed to malathion

The purpose of this study was to evaluate the impact of malathion on the immune system of wild birds, using Japanese quail (Coturnix coturnix japonica) as a model. Quail were exposed to malathion in drinking water at environmentally realistic concentrations (0ppm, 1ppm, and 10ppm). In the fifth week, several arms of the immune response were tested using the T-cell based phytohemagglutinin (PHA) skin test, the B-cell mediated antibody response, and the chemiluminescence assay measuring innate immunity. After the sixth week of malathion exposure, quail were challenged with E. coli O2. The bursa of Fabricius and the spleen were assessed for histopathology. No clinical signs of malathion toxicity were observed. Morbidity or mortality subsequent to E. coli exposure tended (P=0.08) to be higher in the high exposure group (50.0%) compared to the control (22.2%) group. There was no difference in the innate immune response in the malathion exposed birds, however, humoral immunity was suppressed (P=0.03) with the higher malathion exposure. Histopathological evaluation revealed an immunosuppressive effect of malathion on the bursa of Fabricius; bursal atrophy, decreased B-cell density and increased apoptosis in the medulla, and increased connective tissue thickness of the follicular epithelium. Antibody suppression was correlated with bursal changes and peripheral blood lymphocyte count, the organ and cells involved in antibody production. Following the same pattern as other immunotoxicity tests, the PHA T-cell proliferative response also tended to be suppressed in the high exposure group. This study provides evidence that subchronic, moderate malathion exposure is immunotoxic to quail and that testing integrated, functional immunity using an infectious challenge is a better predictor of immunotoxicity than individual responses to immunotoxicity tests. The secondary antibody response, circulating lymphocyte populations, and bursal histopathology were the most sensitive indicators of immune status, as these predicted decreased disease resistance with malathion exposure.

Peptide mimics of peptidoglycan are vaccine candidates and protect mice from infection with Staphylococcus aureus

Staphylococcus aureus drug resistance to antibiotics is a serious situation that has drawn greater attention to immunotherapy and prophylaxis. Peptidoglycan (PGN) is a common and conserved component of the cell wall of Gram-positive bacteria such as S. aureus. However, PGN, as a thymus-independent antigen, cannot be considered a vaccine candidate because of its very weak immunogenicity. In this study we have attempted to enhance the immunogenicity of PGN by identifying a PGN peptide mimic sequence that would act as a thymus-dependent antigen. Several peptide sequences were obtained from a phage display peptide library using a mAb against S. aureus PGN, and a 12-mer linear single peptide (Sp-31) and a four-branch multiple antigen peptide (MAP) (MAP-P31) were synthesized. Both Sp-31 and MAP-P31 were shown to bind directly to anti-PGN mAb and a polyclonal antibody against S. aureus. These peptides could also inhibit the binding of PGN to a mAb against PGN. Furthermore, MAP-P31 was able to provoke an effective secondary antibody response in mice to PGN and to cell-wall fragments isolated from S. aureus, Escherichia coli, Staphylococcus epidermidis and Pseudomonas aeruginosa by sonication. In addition, the MAP-P31 antiserum showed a potent bactericidal or bacteriostatic activity against S. aureus in the presence and absence of complement in vitro. Importantly, immunization with MAP-P31 significantly prolonged the survival and enhanced bacterial clearance in BALB/c mice challenged with live S. aureus. In addition, the serum IgG-type antibodies against PGN persisted in mice, demonstrating that MAP-P31, as a peptide mimicking epitopes on PGN, provokes an effective secondary or memory antibody response, which can only be induced by a thymus-dependent antigen and which protects against infection with S. aureus. These results suggest that MAP-31 may be a novel vaccine candidate against S. aureus.

Recombinant immunotoxin against B-cell malignancies with no immunogenicity in mice by removal of B-cell epitopes

Many nonhuman proteins have useful pharmacological activities, but are infrequently effective in humans because of their high immunogenicity. A recombinant immunotoxin (HA22, CAT8015, moxetumomab pasudotox) composed of an anti-CD22 antibody variable fragment fused to PE38, a 38-kDa portion of Pseudomonas exotoxin A, has produced many complete remissions in drug-resistant hairy-cell leukemia when several cycles of the agent can be given, but has much less activity when antibodies develop. We have pursued a strategy to deimmunize recombinant immunotoxins by identifying and removing B-cell epitopes. We previously reported that we could eliminate most B-cell epitopes using a combination of point mutations and deletions. Here we show the location and amino acid composition of all of the B-cell epitopes in the remaining 25-kDa portion of Pseudomonas exotoxin. Using this information, we eliminated these epitopes to produce an immunotoxin (HA22-LR-8M) that is fully cytotoxic against malignant B-cell lines, has high cytotoxic activity against cells directly isolated from patients with chronic lymphocytic leukemia, and has excellent antitumor activity in mice. HA22-LR-8M does not induce antibody formation in mice when given repeatedly by intravenous injection and does not induce a secondary antibody response when given to mice previously exposed to HA22. HA22-LR-8M also has greatly reduced antigenicity when exposed to sera from patients who have produced antibodies to HA22. The properties of HA22-LR-8M make it an excellent candidate for further clinical development.

Antibody and cell-mediated immune responses and survival between Holstein and Norwegian Red × Holstein Canadian calves

As an extension of a former study, the objectives of this study were to evaluate purebred Holstein (HO; n=140) and crossbred Norwegian Red × Holstein (NRFX; n=142) calves for antibody (AMIR) and cell-mediated immune responses (CMIR) as well as survival. Blood was collected on d 0, 14, and 21, and calves were immunized on d 0 and 14 with type 1 (Candida albicans) and type 2 (hen egg white lysozyme) antigens, which have been shown to induce CMIR and AMIR, respectively. Day 21 background skin-fold measurements of either side of the tail-fold were taken and intradermal injections of test (type 1 antigen) and control (phosphate saline buffer) were administered. Day 23 final skin-fold measurements were taken to assess delayed type hypersensitivity as an indicator of CMIR. Survival data were obtained from CanWest Dairy Herd Improvement. Statistical Analysis System general linear models were used to analyze all immune response and survival data and to determine statistical significance between breeds. Results showed that NRFX had greater primary IgM, IgG, IgG1, and secondary IgG1 antibody response, as well as greater primary IgG1:IgG2 ratio to the type 2 antigen compared with HO. The NRFX also had greater primary IgG1 and IgG2, and secondary IgG2 antibody response as well as greater primary IgG1:IgG2 ratio to the type 1 antigen. The NRFX calves had a tendency toward greater survival from age at immune response testing to calving. No difference was observed between breeds for other secondary antibody response traits or delayed type hypersensitivity. Results indicate NRFX have greater AMIR and therefore may have enhanced defense against extracellular pathogens. This may contribute to increased survival compared with HO. Both breeds, however, likely have similar defense against intracellular pathogens, because no differences in CMIR were observed. In general, these results may suggest that crossbreeding could improve resistance to certain diseases in dairy calves, resulting in decreased input costs to producers for crossbred calves compared with purebred calves. However, more research with larger sample sizes and different breeds should be conducted to confirm these results and obtain a complete picture of the benefits of crossbreeding on immune response traits in calves.

Effects of repeated intratracheally administered lipopolysaccharide on primary and secondary specific antibody responses and on body weight gain of broilers

Earlier, we reported that pathogen-associated molecular patterns such as lipopolysaccharide (LPS), when administered intratracheally (i.t.), affected primary and secondary specific antibody responses to antigens administered concurrently, either i.t. or systemically, and also affected BW gain (BWG) of layers and broilers. In the present study, we evaluated the effects of repeated i.t. challenge with LPS concurrently with or before i.t. immunizations with the specific antigens human serum albumin (HuSA) and rabbit gamma globulin (RGG) on primary (HuSA, RGG) and secondary (HuSA) systemic antibody responses and (isotype) IgM and IgG responses at 2 different ages. Broilers were challenged via the trachea at 3 and 7 wk of age with various combinations of LPS, HuSA, and RGG. All treatments affected immune responses at several time points and also affected BWG, albeit temporarily for the latter. Lipopolysaccharide enhanced primary antibody responses to HuSA and to RGG, when challenged concurrently, but birds challenged solely with LPS at 3 wk of age also showed enhanced primary antibody responses to HuSA and RGG given at 7 wk of age. This was true for IgM as well as IgG isotype responses. Lipopolysaccharide challenge negatively affected BWG at 3 wk of age, whereas the negative effects of LPS after a secondary LPS challenge at 7 wk of age were most pronounced in the birds challenged with LPS at 3 wk of age. The present results indicated that LPS, when administered i.t. at a young age, may affect specific humoral immune responsiveness to antigens administered simultaneously and to BWG of broilers, but also when challenged 4 wk later with specific antigens, suggesting an enhanced status of immune reactivity or sensitivity. The hygienic status of broiler houses at a young age may thus influence BWG, immune responsiveness, and, consequently, the vaccine efficacy and disease resistance in broilers at later ages. The consequences of our findings are discussed.

Chapter five - Vitamin A and Retinoic Acid in the Regulation of B-Cell Development and Antibody Production

Signaling by vitamin A through its active metabolite retinoic acid (RA) is critical for the normal development and functions of the hematopoietic and immune systems. B cells, as both factories for antibody production and part of the immune regulatory system, are critical to a successful vaccination response. RA is a factor in the development and competence of mature B cells, in B cell proliferation, and in the regulation of transcription factors associated with B cell differentiation, class switch recombination, and the generation of antibody-secreting plasma cells. Emerging evidence suggests that RA can function alone and in combination with other immune system stimuli to augment the formation of germinal centers, leading to increased primary and secondary antibody responses. Taken together, RA could be a useful component in vaccine strategies and/or for immunotherapy.

Dedicator of cytokinesis 8 (DOCK8) deficiency.

To describe a new combined primary immunodeficiency disease, previously known as autosomal recessive hyper-IgE syndrome, whose molecular basis was discovered in 2009. Two groups identified homozygous and compound heterozygous loss-of-function mutations in the Dedicator of cytokinesis 8 (DOCK8) gene in at least 30 patients who had been previously diagnosed with an atypical form of hyper-IgE syndrome. Absence of DOCK8 expression impairs T cell expansion in vitro, which could help explain the T cell lymphopenia and susceptibility to cutaneous viral infections observed in these patients. In mouse models of DOCK8 deficiency, absence of DOCK8 expression also impairs the generation of a durable secondary antibody response to specific antigens, which could account for the functional antibody abnormalities and recurrent sinopulmonary infections observed in the patients. Two patients have been cured of infectious complications after myeloablative allogeneic hematopoietic cell transplantation. The discovery of the molecular basis of this disease is expected to facilitate diagnosis and definitive treatment with hematopoietic cell transplantation. Further research is needed to understand how DOCK8 normally functions in lymphocytes and how DOCK8 deficiency leads to disease.

Primary Antibody Responses Against the Novel Pandemic H1N1 and Secondary Antibody Responses Against Seasonal H1N1 Can Be Induced by Vaccination In Patients Early After Allogeneic Stem Cell Transplantation

AbstractAbstract 1273Pandemic influenza (Flu) 2009 and Seasonal Flu are considered a potential health risk for patients after allogeneic stem cell transplantation (alloSCT). However, in a cohort of 137 patients transplanted between October 2006 and December 2009, only 3 patients (2.3%) developed a PCR proven Pandemic Flu infection, and only 1 patient died from multiple super-infections. Moreover, during the Flu season 2009, 5/93 patients (5.3%) transplanted between October 2005 and December 2008 developed a PCR proven Influenza infection and none of them died. These numbers suggest that alloSCT patients were relatively protected against Seasonal and even Pandemic Flu, probably due to earlier Flu antigen exposure. Therefore, we hypothesized that, despite immune impairment after alloSCT, most patients developed secondary and primary antibody responses against Seasonal Flu and neo-antigen containing Pandemic Flu vaccination. To test this hypothesis we measured IgG antibody levels against common Seasonal hemagglutinin (HA), neo-antigens in Pandemic HA and highly conserved cross-reactive re-call antigens shared by Pandemic HA and Seasonal HA in 56 alloSCT patients after vaccination with Seasonal Flu 2009–2010 and Pandemic Flu 2009. To accurately measure antibodies against these antigens, a novel Luminex multiplex bead assay was developed. Recombinant CMV pp65 control protein, native recombinant Seasonal HA (A/Brisbane/59/2007) and Pandemic HA (A/Calefornia/7/2009) were coupled to different Luminex beads which were incubated with plasma samples. To distinguish antibodies against neo-antigenic parts and conserved re-call antigens of Pandemic HA, samples were pre-incubated with or without 0.75 μg HA (A/Brisbane/59/2007) to absorb cross-reacting antibodies against shared parts of Seasonal and Pandemic HA. Finally, samples were stained with PE- labeled Goat α-human IgG and analyzed with a Luminex analyzer. To validate the Luminex bead assay, pp65, Seasonal HA and Pandemic HA positive and negative serum samples were measured with and without pre-incubation with the corresponding protein. In addition, a hemagglutination inhibition assay was performed with Pandemic H1N1 which showed a good correlation with the Luminex assay (r=0.87). Furthermore, specificity of the assay was confirmed by absence of pp65 antibodies in 17 CMV negative patients, whereas in 33/39 (85%) of CMV positive patients antibodies against the immunodominant pp65 protein were detected. To control for vaccination efficacy, 11 healthy donors were vaccinated with Seasonal Flu 2009–2010 followed by 2 Pandemic Flu vaccinations 3 and 6 weeks later. Plasma samples were taken before vaccination, 4 and 12 weeks after Seasonal Flu, and 1 and 8 weeks after Pandemic Flu vaccination. Prior to vaccination all donors had antibodies against Seasonal HA and conserved re-call Pandemic HA, reflecting their previous vaccination with Seasonal Flu 2008–2009. All donors developed secondary antibody responses against Seasonal HA. Highly specific Pandemic HA specific antibodies were present in 1 donor before and 10/11 donors after Pandemic Flu vaccination, indicating that 9/11 donors developed a primary antibody response to neo-antigenic Pandemic HA. To investigate primary and secondary responses after Seasonal and Pandemic Flu vaccination in alloSCT patients, 56 patients followed a similar vaccination and plasma donation scheme. 86% had antibodies against Seasonal HA and 76% against conserved Pandemic HA pre vaccination, probably induced by vaccinations before 2009. 55% developed a secondary antibody response against Seasonal HA 4 –12 weeks post vaccination. Only 5% had neo-antigenic Pandemic HA antibodies prior to vaccination. Excitingly, 57% developed a primary antibody response against neo-antigenic Pandemic HA 8 weeks after Pandemic Flu vaccination. Overall, patients received vaccinations at a median of 443 (range 90–1240) days after alloSCT. Patients vaccinated 90–300, 301–600 and >600 days after alloSCT developed primary antibody responses against neo-antigenic Pandemic HA in 41%, 47% and 80% of the cases, respectively. In conclusion, this detailed analysis of primary and secondary antibody responses against re-call and neo-antigens after Seasonal and 2009 Pandemic HA may explain the low incidence of Flu infections in patients after alloSCT and provides rational arguments to start Flu vaccination from 3 months after alloSCT. Disclosures:No relevant conflicts of interest to declare.

ANTIBODY RESPONSES FOLLOWING SPLENECTOMY: IMPLICATIONS FOR THE TIMING OF PROPHYLACTIC VACCINATION

Antibody responses are reduced both in the short- and long-term after splenectomy. It is not known if this reduction is more profound in the immediate postoperative period compared to later, and consequently, whether there would be an advantage in delaying prophylactic vaccination to ensure a greater antibody response. To investigate this, the effect of splenectomy on the primary and secondary antibody response to sheep red blood cells (SRBC) injected into the peritoneum (IP) of adult male Porton rats was measured after either splenectomy (Spx), sham splenectomy (Sham), anaesthesia only (Ans) or no procedure at all (Con). There was no difference in the titres of antibody between the Con and the Ans rats. There was no difference in the titres of antibody between the Sham and Spx rats, but both had significantly lower titres than the Con rats. In a separate experiment, rats were immunized 1 month or 1 year after splenectomy or sham-splenectomy. One month after the operation the antibody response of the Sham rats had increased and did not differ from that of Con rats, but in rats which had been splenectomized there was a long-term suppression of the antibody response, which did not improve for at least one year. The results suggest that, in patients requiring splenectomy for trauma and not able to be vaccinated before operation, there may be no advantage in delaying prophylactic vaccination postoperatively.

Unexpected modulation of recall B and T cell responses after immunization with rotavirus-like particles in the presence of LT-R192G.

LT-R192G, a mutant of the thermolabile enterotoxin of E. coli, is a potent adjuvant of immunization. Immune responses are generally analyzed at the end of protocols including at least 2 administrations, but rarely after a prime. To investigate this point, we compared B and T cell responses in mice after one and two intrarectal immunizations with 2/6 rotavirus-like particles (2/6-VLP) and LT-R192G. After a boost, we found, an unexpected lower B cell expansion measured by flow cytometry, despite a secondary antibody response. We then analyzed CD4+CD25+Foxp3+ regulatory T cells (Tregs) and CD4+CD25+Foxp3− helper T cells after in vitro (re)stimulation of mesenteric lymph node cells with the antigen (2/6-VLP), the adjuvant (LT-R192G) or both. 2/6-VLP did not activate CD4+CD25+Foxp3− nor Foxp3+ T cells from non-immunized and 2/6-VLP immunized mice, whereas they did activate both subsets from mice immunized with 2/6-VLP in the presence of adjuvant. LT-R192G dramatically decreased CD4+CD25+Foxp3+ T cells from non-immunized and 2/6-VLP immunized mice but not from mice immunized with 2/6-VLP and adjuvant. Moreover, in this case, LT-R192G increased Foxp3 expression on CD4+CD25+Foxp3+ cells, suggesting specific Treg activation during the recall. Finally, when both 2/6-VLP and LT-R192G were used for restimulation, LT-R192G clearly suppressed both 2/6-VLP-specific CD4+CD25+Foxp3− and Foxp3+ T cells. All together, these results suggest that LT-R192G exerts different effects on CD4+CD25+Foxp3+ T cells, depending on a first or a second contact. The unexpected immunomodulation observed during the recall should be considered in designing vaccination protocols.