High Ab Levels Research Articles

Novel Bivalent Viral-Vectored Vaccines Induce Potent Humoral and Cellular Immune Responses Conferring Protection against Stringent Influenza A Virus Challenge.

Seasonal influenza viruses are a common cause of acute respiratory illness worldwide and generate a significant socioeconomic burden. Influenza viruses mutate rapidly, necessitating annual vaccine reformulation because traditional vaccines do not typically induce broad-spectrum immunity. In addition to seasonal infections, emerging pandemic influenza viruses present a continued threat to global public health. Pandemic influenza viruses have consistently higher attack rates and are typically associated with greater mortality compared with seasonal strains. Ongoing strategies to improve vaccine efficacy typically focus on providing broad-spectrum immunity; although B and T cells can mediate heterosubtypic responses, typical vaccine development will augment either humoral or cellular immunity. However, multipronged approaches that target several Ags may limit the generation of viral escape mutants. There are few vaccine platforms that can deliver multiple Ags and generate robust cellular and humoral immunity. In this article, we describe a novel vaccination strategy, tested preclinically in mice, for the delivery of novel bivalent viral-vectored vaccines. We show this strategy elicits potent T cell responses toward highly conserved internal Ags while simultaneously inducing high levels of Abs toward hemagglutinin. Importantly, these humoral responses generate long-lived plasma cells and generate Abs capable of neutralizing variant hemagglutinin-expressing pseudotyped lentiviruses. Significantly, these novel viral-vectored vaccines induce strong immune responses capable of conferring protection in a stringent influenza A virus challenge. Thus, this vaccination regimen induces lasting efficacy toward influenza. Importantly, the simultaneous delivery of dual Ags may alleviate the selective pressure that is thought to potentiate antigenic diversity in avian influenza viruses.

PspA-based sublingual vaccine provides IgA dependent protective immunity to nasopharyngeal carriage by Streptococcus pneumoniae

Abstract Pneumococcal infection caused by Streptococcus pneumoniae is a major upper respiratory tract disease which causes severe illness and mortality. It is important to develop safe and effective vaccines to prevent pneumococcal infections. Sublingual (SL) antigen (Ag) delivery is a non-invasive route which avoids possible entry into the olfactory region and the central nervous system. We examined the potential of Poly (I:C) as a SL adjuvant for the induction of specific immune responses against S. pneumoniae infection. C57BL/6 and IgA−/− mice were SL immunized with 1 μg of pneumococcal surface protein A (PspA) and 10 μg of Poly (I:C) four times at weekly intervals. One week after the final immunization, nasal washes (NWs), saliva and plasma samples were collected and subjected to a PspA-specific ELISA. Further, mice were challenged with S. pneumoniae strain EF3030, and the numbers of CFUs in NWs and nasal passages (NPs) were determined. Significantly increased levels of PspA-specific secretory IgA (SIgA) antibody (Ab) responses were seen in NWs of mice given SL PspA plus Poly (I:C) when compared with controls. Further, higher levels of PspA-specific IgG and IgA Abs were noted in plasma of vaccinated mice. Importantly, mice given SL PspA plus Poly (I:C) showed significantly lower numbers of bacteria in the NPs and NWs when compared with controls. Although IgA−/− mice given SL PspA-vaccine had significantly high levels of PspA-specific IgG Abs, high numbers of CFUs were detected in NWs and NP. These results show that SL immunization is effective mucosal immunization strategy for induction of protective pneumococcal-specific Ab responses and specific SIgA Abs play a necessary role in the blockade of S. pneumoniae colonization of the nasal cavity.

Single N277A substitution in C2 of simian immunodeficiency virus envelope influences vaccine-elicited CD4i neutralizing and anti-V2 antibody responses

An effective HIV vaccine remains elusive, and immunogens capable of eliciting protective host humoral immunity have not yet been identified. Although HIV/SIV infections result in the abundant production of CD4-induced (CD4i) antibodies (Abs), these Abs are not protective due to steric restrictions following gp120 binding to CD4 on target cells. Here we report that both DNA- and vaccinia-based vaccines encoding SIVmac239 gp160 readily elicited high levels of CD4i Abs in experimental animals. We identified a highly conserved N-linked glycosylation site N277 in the C2 region which strongly affected the immunogenicity of the CD4i Ab domain. Moreover, a single N277A substitution significantly enhanced the immunogenicity of the V2 domain yielding higher titers and frequency of anti-V2 Ab responses as determined by ELISA and yeast antigen display mapping, respectively. Importantly, immune sera elicited by the N277A-mutated gp160 exhibited elevated antibody-dependent cellular cytotoxicity (ADCC) activity. ADCC activity correlated positively with the anti-V2 Ab titer yet, inversely with CD4i Ab titer. Thus, we identified a determinant of the CD4i domain that might affect vaccine-elicited anti-V2 Ab and ADCC responses to SIVmac239. Our findings may have implications for design of immunogens to direct B cell recognition in the development of an Ab-based HIV vaccine.

Analysis of Pre-existing IgG and IgM Antibodies against Polyethylene Glycol (PEG) in the General Population.

Circulating antibodies (Ab) that specifically bind polyethylene glycol (PEG), a biocompatible polymer routinely used in protein and nanoparticle therapeutics, have been associated with reduced efficacy of and/or adverse reactions to therapeutics modified with or containing PEG. Unlike most antidrug antibodies that are induced following initial drug dosing, anti-PEG Ab can be found in treatment-naïve individuals (i.e., individuals who have never undergone treatment with PEGylated drugs but most likely have been exposed to PEG through other means). Unfortunately, the true prevalence, quantitative levels, and Ab isotype of pre-existing anti-PEG Ab remain poorly understood. Here, using rigorously validated competitive ELISAs with engineered chimeric anti-PEG monoclonal Ab standards, we quantified the levels of anti-PEG IgM and different subclasses of anti-PEG IgG (IgG1-4) in both contemporary and historical human samples. We unexpectedly found, with 90% confidence, detectable levels of anti-PEG Ab in ∼72% of the contemporary specimens (18% IgG, 25% IgM, 30% both IgG and IgM). The vast majority of these samples contained low levels of anti-PEG Ab, with only ∼7% and ∼1% of all specimens possessing anti-PEG IgG and IgM in excess of 500 ng/mL, respectively. IgG2 was the predominant anti-PEG IgG subclass. Anti-PEG Ab's were also observed in ∼56% of serum samples collected during 1970-1999 (20% IgG, 19% IgM, and 16% both IgG and IgM), suggesting that the presence of PEG-specific antibodies may be a longstanding phenomenon. Anti-PEG IgG levels demonstrated correlation with patient age, but not with gender or race. The widespread prevalence of pre-existing anti-PEG Ab, coupled with high Ab levels in a subset of the population, underscores the potential importance of screening patients for anti-PEG Ab levels prior to administration of therapeutics containing PEG.

Annexin A2, autoimmunity, anxiety and depression

ObjectivesAntiphospholipid syndrome (APS) is associated with neurological manifestations and one of the novel autoantigens associated with this disease is Annexin A2 (ANXA2). In this work we have examined the effect of high levels of autoantibodies to ANXA2 on the brain in a mouse model. MethodsRecombinant ANXA2 emulsified in adjuvant was used to immunize mice while mice immunized with adjuvant only served as controls. At peak antibody levels the animal underwent behavioral and cognitive tests and their brains were examined for ANXA2 immunoglobulin G (IgG) and expression of ANXA2 and the closely linked protein p11. ResultsVery high levels of anti-ANXA2 antibodies (Abs) were associated with reduced anxiety in the open field 13.14% ± 0.89% of the time in the center compared to 8.64% ± 0.91% observed in the control mice (p < 0.001 by t-test). A forced swim test found significantly less depression manifested by immobility in the ANXA2 group. The changes in behavior were accompanied by a significant reduction in serum corticosteroid levels of ANXA2 group compared to controls. Moreover, higher levels of total IgG and p11 expression were found in ANXA2 group brains. Lower levels of circulating anti-ANXA2 Abs were not associated with behavioral changes. ConclusionsWe have established an animal model with high levels of anti-ANXA2 Abs which induced IgG accumulation in the brain and specific anxiolytic and anti-depressive effects. This model promises to further our understanding of autoimmune disease such as APS and to provide better understanding of the role of the ANXA2-p11 complex in the brain.

Generation of Catalytic Antibodies Is an Intrinsic Property of an Individual's Immune System: A Study on a Large Cohort of Renal Transplant Patients.

Renal transplant is the treatment of choice for patients with terminal end-stage renal disease. We have previously identified low levels of catalytic IgG as a potential prognosis marker for chronic allograft rejection. The origin and physiopathological relevance of catalytic Abs is not well understood, owing to the fact that catalytic Abs have been studied in relatively small cohorts of patients with rare diseases and/or without systematic follow-up. In the current study, we have followed the evolution of the levels of catalytic IgG in a large cohort of renal transplant patients over a 2-y period. Our results demonstrate that, prior to transplant, patients with renal failure present with heterogeneous levels of IgG hydrolyzing the generic proline-phenylalanine-arginine-methylcoumarinamide (PFR-MCA) substrate. PFR-MCA hydrolysis was greater for patients' IgG than for a therapeutic preparation of pooled IgG from healthy donors. Renal transplant was marked by a drastic decrease in levels of catalytic IgG over 3 mo followed by a steady increase during the next 21 mo. Patients who displayed high levels of catalytic IgG pretransplant recovered high levels of catalytic Abs 2 y posttransplant. Interestingly, IgG-mediated hydrolysis of a model protein substrate, procoagulant factor VIII, did not correlate with that of PFR-MCA prior transplantation, whereas it did 12 mo posttransplant. Taken together, our results suggest that the level of circulating catalytic IgG under pathological conditions is an intrinsic property of each individual's immune system and that recovery of pretransplant levels of catalytic IgG is accompanied by changes in the repertoire of target Ags.

PD-1 inhibition results in reduced dendritic cell (DC) activation of HIV-1-specific de novo CTL and enhancement of DC-induced CTL memory recall responses

Abstract Combination strategies consisting of DC vaccines and suppression of immunoregulatory pathways such as PD-1 blockade have been advocated to elicit optimal CTL responses in chronic diseases including cancer and HIV infection. We have shown that inactivated HIV-1 virus loaded, high IL-12 producing, mature, type-1 polarized DC (DC1) can activate CTL from naïve CD8+ T cell precursors that more effectively kill HIV-1-infected cells than CTL derived from memory CD8+ T cells. Here we tested DC1 transfected with an adenoviral (Ad) vector encoding anti-PD-1 antibody (DC.αPD1) enhancement of both primary as well as memory CTL responses to HIV-1. Ad-αPD1-transduced DC1 (DC.αPD1) secreted high levels of functional anti-PD1 Ab without affecting their phenotype or IL-12p70-producing capacity. We next compared HIV Gag peptide epitope-loaded DC.αPD1 to DC1 that were transfected with an empty vector for their ability to activate either purified, autologous naïve or memory HIV-1-specific CD8+ T cells from chronic HIV-1-infected participants of the Multicenter AIDS Cohort Study. When compared to the control DC1, antigen-loaded DC.αPD1 enhanced the overall magnitude of HIV-1-specific CTL responses induced, as determined by the expansion of HIV-1-peptide responsive CD107a and IFNγ expressing T cells. In contrast, the overall number HIV-1 antigen-reactive CTL derived from the naïve CD8+ T cell fraction sharply decreased when using the DC.αPD1-based approach. These results suggest previously unrecognized, opposing roles of the inhibitory receptor PD-1 in DC1-induced primary versus memory recall CD8+ T cell responses to HIV-1.

Bovine Coronavirus (BoCV) Infection in Calves with Diarrhoea and Their Dams

Background: Bovine coronavirus (BoCV) is common with high seroprevalence in dairy cattle. It is reported in many countries. Also, BoCV causes diarrhea in dairy calves. The transmission of BoCV is the fecal-oral/aerosol-nasal routes. Feces from clinical cases or clinically normal dairy cattle are source of infection, also contamination of feed and water. The purpose of the current study was to investigate BoCV infection in diarrheic calves (age and sex) and their dams (age). For this reason, the serological and virological methods were used. Haematological parameters of the calves and their dams were compared using the statistical methods.Materials, Methods &amp; Results: In this study, following clinical examination of 3500 cattle and their calves from 25 number of dairy farms 184 calves with diarrhoea and their dams (183) (≥2 - ≤6 age) were sampled for BoCV presence by enzyme linked immunosorbent assay (ELISA). Additionally, all blood samples were examined by hematological methods. 172 (93.99%) cows and 172 (93.99%) calves were found antibodies (Ab) positive (+). The high levels of Ab for BoCV were detected as 36.05 % in dams 6 years and older ages. In the calves, Ab to BoCV were found at the highest level (25.26%) in the female calves ≥5 - ≤6 months ages. BoCV antigen (Ag) was detected in only faecal sample of a (0.54%) calf. When the haematological parameters were compared between BoCV Ab (+) and BoCV Ab negative (-) dams, only white blood cell (WBC) values were found statistically significant (P &lt; 0.05). When the haematological parameters were compared between BoCV Ab (+)/Ag (-) and BoCV Ab (-)/Ag (-) calves, WBC (P &lt; 0.05), lymphocyte (P &lt; 0.01) and granulocyte (P &lt; 0.01) values were found statistically important. When the haematological parameters were compared between BoCV Ab (+)/Ag (+) and BoCV Ab (-)/Ag (-) calves, both lymphocyte and granulocyte values were statistically important (P &lt; 0.01).Discussion: BoCV infection has found worldwide among cattle of all ages. The disease results in major economic losses in dairy herds that result from treatment costs and calf deaths. One hundred seventy two out of 183 mothers whose blood sampling was done were detected as seropositive. Many researchers found similar results in dairy cattle. It was detected the highest seropositivity in cattle more than six years old. One hundred seventy two blood samples out of 184 calves were detected seropositive. Also, the highest seropositivity was detected among of &gt; 5 and &lt; 6 months of age. BoCV Ag (+) presence was detected in only one faecal sample of one calf out of 184. Researchers were found same or higher BoCV Ag(+) rates in faeces of diarrheic calves. In the study lymphocyte counts of seropositive cows and in Ab(+)/Ag(+) calves determined decrease. However, the counts in seropositive calves were increased. Leukocytes levels were also high in seropositive calves. Haematocrit values were decreased in seropositive cows, calves and in Ab(+)/Ag(-) calves. BoCV infections were detected at low level in diarrheic calves. But, BoCV seropositive mature and diarrheic calves were found at high levels. Haematological application methods could be used to be supportive with the serological and virological methods. All farm managements should be maintained with strict hygiene practices. Milking bottle, calf pens or hutches need to be sanitized. The calves must be prevent contamination from faeces and urine of other calves. The protective vaccination must be applied all animals.

Mechanisms of B Cell Tolerance after in Utero Hematopoietic Cell Transplantation

Background: In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative, nonimmunosuppressive transplant approach that results in donor cell engraftment across immune barriers. Although a significant amount of work has investigated the fate of T cells following IUHCT, little attention has been paid to B cell tolerance and the fate of donor derived host reactive or host derived donor reactive B cells following IUHCT. B cell tolerance is broadly believed to occur by a combination of 3 mechanisms: deletion, receptor editing, and functional inactivation (anergy). In the current study we attempt to elucidate the mechanism(s) by which B cell tolerance occurs following IUHCT.Methods: 10x106 donor bone marrow (BM) cells were injected intravenously via the vitelline vein into gestational day 14 murine fetuses. IUHCT was performed in the congenic (C57Bl/6-GFP [H2Kb ] into C57Bl/6 [H2Kb ]) and allogeneic (C57Bl/6-GFP into Balb/c [H2Kd ]) strain combinations. Naive Balb/c and C57Bl/6 mice served as controls. Mice were sacrificed at day of life 3 (P3), 1 month and 4 months of age at which time their BM, spleen, and serum were harvested. To assess B cell deletion, flow cytometry was used to determine the absolute # and % of host and donor immature and pre B cells in the BM. Additionally, apoptosis of host and donor BM derived B cells was determined by annexin staining. Central receptor editing was evaluated using RT qPCR to measure the amount of Vκ-RS rearrangments in BM pre-B cells. Peripheral receptor editing was studied by calculating the % of λ light chains in mature splenocytes identified by flow cytometry. Finally, functional inactivation of donor reactive host B cells was assessed by measuring anti-H2Kb serum antibodies (ab) of allogeneic chimeras, naive, and immunized mice at 1 month of age.Results: The absolute number of BM immature B cells was decreased in allogeneic recipients of IUHCT compared to noninjected Balb/c controls at 1 month of age (fig 1). This effect was lost by 4 months of age. The decrease in B cells resulted primarily from a decrease in immature donor as opposed to host B cells compared to controls (% immature donor B cells in allogeneic recipients vs. controls: 16.2% vs. 39.9%; p<0.0005). Donor B cells in allogeneic chimeras also demonstrated a trend toward increased apoptosis compared to controls (24.8 vs. 18.7%; p=0.2) which was not seen in immature host B cells (18.3 vs. 18.6%; p=0.9). There was no significant decrease in the absolute number of immature B cells or increased apoptosis in congenic recipients compared to uninjected controls. These findings suggest deletion of autoreactivedonor B cells.Light chain receptor editing involves rearrangements within the κ and λ gene loci and may occur in BM pre-B cells or mature B cells in the spleen. We found no difference in the Vκ-RS rearrangements of pre B cells in allogeneic chimeras and controls at 1 month. In contrast, the quantity of total λ+ mature splenic B cells was increased in allogeneic chimeras at P3 (10.8 vs. 8.4%; p=0.02) and resulted from an increased host λ+ % compared to controls (10.8 vs. 8.4% p=0.03) suggesting peripheral receptor editing of host cells (fig.2). The λ+ % increase in allogeneic chimeras was lost by 1 month.Autoreactive B cells that escape deletion and receptor editing can be functionally inactivated. Neither allogeneic nor naive mice developed ab to H2Kb splenocytes, however, Balb/c mice immunized to H2kb antigen showed high ablevels (MFI fold change: allo-0.89 naive-1.37 imm-2.77; p<0.05).Conclusion:B cell tolerance after IUHCT is achieved by distinct mechanisms for host and donor cell populations. Donor derived host reactive B cells undergo deletion and apoptosis while receptor editing and functional inactivation are the primary mechanisms of B cell tolerance of host derived donor reactive B cells. We hope use this and future studies of antigen specific B cell tolerance to harness the immunologic potential of IUHCT for many hematopoietic and immunologic congenital diseases. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

P146 Establishing a sexual health research practice network in the North East

Seasonal influenza viruses are a common cause of acute respiratory illness worldwide and generate a significant socioeconomic burden. Influenza viruses mutate rapidly, necessitating annual vaccine reformulation because traditional vaccines do not typically induce broad-spectrum immunity. In addition to seasonal infections, emerging pandemic influenza viruses present a continued threat to global public health. Pandemic influenza viruses have consistently higher attack rates and are typically associated with greater mortality compared with seasonal strains. Ongoing strategies to improve vaccine efficacy typically focus on providing broad-spectrum immunity; although B and T cells can mediate heterosubtypic responses, typical vaccine development will augment either humoral or cellular immunity. However, multipronged approaches that target several Ags may limit the generation of viral escape mutants. There are few vaccine platforms that can deliver multiple Ags and generate robust cellular and humoral immunity. In this article, we describe a novel vaccination strategy, tested preclinically in mice, for the delivery of novel bivalent viral-vectored vaccines. We show this strategy elicits potent T cell responses toward highly conserved internal Ags while simultaneously inducing high levels of Abs toward hemagglutinin. Importantly, these humoral responses generate long-lived plasma cells and generate Abs capable of neutralizing variant hemagglutinin-expressing pseudotyped lentiviruses. Significantly, these novel viral-vectored vaccines induce strong immune responses capable of conferring protection in a stringent influenza A virus challenge. Thus, this vaccination regimen induces lasting efficacy toward influenza. Importantly, the simultaneous delivery of dual Ags may alleviate the selective pressure that is thought to potentiate antigenic diversity in avian influenza viruses.

P. falciparum and P. vivax Epitope-Focused VLPs Elicit Sterile Immunity to Blood Stage Infections.

In order to design P. falciparum preerythrocytic vaccine candidates, a library of circumsporozoite (CS) T and B cell epitopes displayed on the woodchuck hepatitis virus core antigen (WHcAg) VLP platform was produced. To test the protective efficacy of the WHcAg-CS VLPs, hybrid CS P. berghei/P. falciparum (Pb/Pf) sporozoites were used to challenge immunized mice. VLPs carrying 1 or 2 different CS repeat B cell epitopes and 3 VLPs carrying different CS non-repeat B cell epitopes elicited high levels of anti-insert antibodies (Abs). Whereas, VLPs carrying CS repeat B cell epitopes conferred 98% protection of the liver against a 10,000 Pb/Pf sporozoite challenge, VLPs carrying the CS non-repeat B cell eptiopes were minimally-to-non-protective. One-to-three CS-specific CD4/CD8 T cell sites were also fused to VLPs, which primed CS-specific as well as WHcAg-specific T cells. However, a VLP carrying only the 3 T cell domains failed to protect against a sporozoite challenge, indicating a requirement for anti-CS repeat Abs. A VLP carrying 2 CS repeat B cell epitopes and 3 CS T cell sites in alum adjuvant elicited high titer anti-CS Abs (endpoint dilution titer >1x106) and provided 80–100% protection against blood stage malaria. Using a similar strategy, VLPs were constructed carrying P. vivax CS repeat B cell epitopes (WHc-Pv-78), which elicited high levels of anti-CS Abs and conferred 99% protection of the liver against a 10,000 Pb/Pv sporozoite challenge and elicited sterile immunity to blood stage infection. These results indicate that immunization with epitope-focused VLPs carrying selected B and T cell epitopes from the P. falciparum and P. vivax CS proteins can elicit sterile immunity against blood stage malaria. Hybrid WHcAg-CS VLPs could provide the basis for a bivalent P. falciparum/P. vivax malaria vaccine.

Prevalence study and genetic typing of bovine viral diarrhea virus (BVDV) in four bovine species in China.

To determine the nationwide status of persistent BVDV infection in different bovine species in China and compare different test methods, a total of 1379 serum samples from clinical healthy dairy cattle, beef cattle, yaks (Bos grunniens), and water buffalo (Bubalus bubalis) were collected in eight provinces of China from 2010 to 2013. The samples were analyzed using commercial antibody (Ab) and antigen (Ag) detection kits, and RT-PCR based on the 5’-UTR and Npro gene sequencing. Results showed that the overall positive rates for BVDV Ab, Ag and RT-PCR detection were 58.09% (801/1379), 1.39% (14/1010), and 22.64% (146/645), respectively, while the individual positive rates varied among regions, species, and farms. The average Ab-positive rates for dairy cattle, beef cattle, yaks, and water buffalo were 89.49% (298/333), 63.27% (248/392), 45.38% (236/520), and 14.18% (19/134), respectively, while the Ag-positive rates were 0.00% (0/116), 0.77% (3/392), 0.82% (3/368), and 5.97% (8/134), respectively, and the nucleic acid-positive rates detected by RT-PCR were 32.06% (42/131), 13.00% (26/200), 28.89% (52/180), and 19.40% (26/134), respectively. In addition, the RT-PCR products were sequenced and 124 5’-UTR sequences were obtained. Phylogenetic analysis of the 5’-UTR sequences indicated that all of the 124 BVDV-positive samples were BVDV-1 and subtyped into either BVDV-1b (33.06%), BVDV-1m (49.19%), or a new cluster, designated as BVDV-1u (17.74%). Phylogenetic analysis based on Npro sequences confirmed this novel subtype. In conclusion, this study revealed the prevalence of BVDV-1 in bovine species in China and the dominant subtypes. The high proportion of bovines with detectable viral nucleic acids in the sera, even in the presence of high Ab levels, revealed a serious threat to bovine health.

Parainfluenza virus type 3 Ab in allogeneic hematopoietic cell transplant recipients: factors influencing post-transplant Ab titers and associated outcomes.

Parainfluenza virus type 3 (PIV-3) can cause severe respiratory illness among hematopoietic cell transplantation (HCT) recipients. Factors associated with PIV-3-specific Ab level, and the association between PIV-3 Ab levels and clinical outcomes in HCT recipients who acquire PIV-3 infection, are unknown. We evaluated PIV-3-specific hemagglutination inhibition Ab levels and clinical outcomes among 172 patients with PIV-3 infection following HCT. In a multivariable linear regression model, high post-transplantation Ab levels were independently associated with higher pre-transplantation recipient titer (mean difference 0.38 (95% confidence interval (CI), 0.26, 0.50), P<0.001). Significant associations between pre-HCT Ab titers in both patients and donors and occurrence of lower respiratory tract disease (LRD) after HCT were not observed. In conclusion, low pre-transplantation titers are associated with low Ab levels after HCT. The relationship between PIV-3 Ab levels and outcomes remain uncertain. Further study is needed to prospectively evaluate the dynamics of PIV-3-specific Ab responses and the relative contribution of PIV-3-specific Ab to protection from infection acquisition and progression to LRD.

Interleukin-6 promotes systemic lupus erythematosus progression with Treg suppression approach in a murine systemic lupus erythematosus model.

Our aim is to reveal the role of interleukin 6 (IL-6) in the pathogenesis of systemic lupus erythematosus (SLE) in a murine model of SLE. Normal female C57BL/6 mice were immunized with syngeneic-activated lymphocyte-derived DNA (ALD-DNA) to induce SLE. Non-immunized mice were used as control. SLE-associated markers, including anti-double-stranded DNA (anti-dsDNA) Abs, urine protein, and kidney histopathology, were assayed to ensure the induction of the disease. Compared with control mice, ALD-DNA immunized mice exhibited high levels of anti-dsDNA Abs, IL-6 expression in vivo and in vitro. We also found that IL-6 knockout (IL-6KO) mice were resistant to ALD-DNA-induced SLE. The activation of CD4(+) T cells in immunized IL-6KO mice was lower than in immunized wild-type (Wt) mice. Intracellular cytokine staining showed that Foxp3 expression in immunized IL-6KO mice was higher than in immunized Wt mice, which might be associated with the disease severity. We further discovered that ALD-DNA-stimulated dendritic cells supernatants could result in higher IL-6 and TNF-α expression and could suppress Foxp3 expression. In addition, blocking IL-6 could up-regulate Foxp3 expression. Therefore, our findings show that IL-6 promotes the progression of SLE via suppressing Treg differentiation.

Evolution of autoantibodies profile in systemic lupus erythematosus according to age and clinical manifestations

Clinical features and auto-antibodies profile of 35 Senegalese patients' diagnosed systemic lupus erythematosus (SLE) were analyzed after measurement of antinuclear antibodies (ANA) by IFI, detection of Abs anti-DNA native by ELISA and evaluation of antibodies anti-Sm, anti-RNP, anti-SSA anti-SSB, anti-CCP2, anti-J0, and anti-Scl70 levels by immunodot. Mean age of 33 yrs (18-50 yrs) and sex ratio (F/M) of 16 were found. The most frequent clinical features were rheumatic (88.7%) and cutaneous (79.4%) disorders. ANA and anti-DNAn Abs were detected in 85.7% and 62.5% of the patients respectively. Abs anti-RNP, anti-Sm, anti-SSA, anti-SSB and anti-CCP2 were detected in 30 to 70% of patients. In young patients, the levels of anti-DNAn and anti-Sm Abs were higher than in patients older than 40 yrs (P<0.05). In addition, associations of cutaneous and rheumatic symptoms were characterized by high levels of anti-DNAn, anti-SSA and anti-SSB Abs. Our study shows the interest of a measurement of anti-DNAn, anti-SSA and anti-SSB Abs during the follow of SLE patients particularly in those presenting both rheumatic and cutaneous symptoms.

Tolerogenic microenvironment in neonatal period induced by maternal immunization with ovalbumin

Maternal immunization with allergens, such as ovalbumin (OVA), can inhibit the development of an allergic response in offspring. The regulatory mechanisms seem to be mediated by maternal antibodies (MatAbs) and factors generated by the maternal–fetal interface. The aim of this study was to verify the pathways of inhibitory Ab transference after maternal immunization with OVA and the effect of the offspring's dendritic cells (DCs) on the generation of regulatory T (Treg) cells. We verified that preconceptional OVA immunization induces high levels of proinflammatory and regulatory cytokines in the amniotic fluid, allowing the transference of high levels of anti-OVA IgG1 Abs to the offspring. Using an adoptive nursing protocol, we verified that maternal immunization leads to MatAb transference by the placental route and by breastfeeding contribute to the inhibition of anaphylactic IgE and IgG1 Ab responses in immunized offspring. We observed that maternal immunization decreased eosinophil numbers in recovered bronchoalveolar lavage fluid and in the lung tissue, whereas with a lack of control of airway responsiveness to methacholine. Maternal immunization induced in young offspring a decreased percentage of CD11c+ DCs expressing MHC class II and CD40 molecules. Moreover, DCs from both groups of offspring when pulsed with OVA, were able to induce Treg cells in vitro. Similarly, OVA immunization at the neonatal stage increased the frequency of Treg cells, regardless of the mother's immunization status. These findings emphasize that maternal immunization leads to a complex interaction of regulatory factors, with MatAbs, DCs and Treg cells affecting the tolerance of offspring during an allergic response.

The Addition Of Interleukin-6 Inhibition To Standard Gvhd Prophylaxis Prevents Acute Gvhd: Interim Results Of a Phase I/II Clinical Study

We and others have demonstrated the dysregulation of interleukin-6 (IL-6) early after experimental bone marrow transplantation (BMT) and protection from acute GVHD following the administration of an anti-IL-6 receptor (IL-6R) antibody. In these models, where GVHD prophylaxis is not administered, systemic IL-6, IFNγ and TNF levels peak 7 days after BMT before returning to baseline by the third week. We have determined cytokine dysregulation in a large clinical cohort of allogeneic stem cell transplant (SCT) recipients conditioned with myeloablative Cy/TBI (12 Gy, n = 25) or reduced intensity Flu/Mel (120mg/m2, n = 25) receiving standard GVHD prophylaxis with cyclosporine and MTX (d 1 at 15mg/m2, d 3, 6, 11 at 10mg/m2). IL-6 levels rose from pre-transplant levels of 6.4 ± 0.7 pg/ml to a peak of 58.8 ± 8.8 pg/ml at day 7 (P < 0.0001) with a fall at day 14 to 39.0 ± 12.5 pg/ml (P < 0.0001) and return to baseline by day 30 (6.2 ± 0.9 pg/ml), consistent with the preclinical data. IL-6 dysregulation was not different in recipients of matched sibling or unrelated donor grafts but was proportional to the intensity of conditioning (day 7 levels after Cy/TBI vs. Flu/Mel: 83.3 ± 12.2 pg/ml vs. 31.0 ± 10.1 pg/ml, P< 0.0001). In contrast to preclinical mouse data, no systemic increases were seen in any other cytokine including IFNγ, TNF, IL-17, IL-4, IL-13 and IL-10.We thus initiated a phase I/II study whereby a human neutralizing monoclonal antibody (mAb) against the IL-6R was administered on day -1 to patients receiving Cy/TBI or Flu/Mel conditioned allogeneic SCT from HLA (10/10)–matched sibling or unrelated donors with standard cyclosporine/MTX GVHD prophylaxis. There was no T cell depletion. The primary endpoint was the incidence of grade II-IV acute GVHD and the study has achieved its planned enrollment (n = 48). There was no toxicity attributable to IL-6R antibody administration. Pharmacokinetic analysis confirmed high levels of IL-6R Ab at day 3 (mean 64.7 ug/ml) which persisted in all patients 3 weeks after BMT (mean = 9.8 ug/ml) and remained above the level of detection (0.1ug/ml) in 75% of patients at day 30 (mean = 1.9 ug/ml). IL-6 levels were dramatically increased (relative to baseline) in patients receiving antibody due to the inability to excrete the inactive IL-6 – soluble IL-6R antibody complex (peak IL-6 levels at day 7 = 773.6 ± 207.9 pg/ml; P < 0.0001) and remained increased at day 30 (60.9 ± 24.4 pg/ml; P < 0.0001), returning to baseline by day 60 (9.5 ± 1.7 pg/ml), consistent with antibody clearance. Soluble IL-6R levels also rose over the first month of SCT and levels at day 30 correlated with residual antibody levels (r2 = 0.72, P = 0.02). Neutrophil (> 0.5x109/L) and platelet (> 20x109/L) recovery was normal relative to a matched untreated control cohort at a median of 16 and 18 days respectively. Donor chimerism and immune reconstitution (conventional T, regulatory T and B cells) was equivalent at day 30 in recipients of IL-6R inhibition versus the control cohort. In contrast, changes in innate immunity were seen in patients receiving IL-6R inhibition with increases in plasmacytoid DC (P = 0.002), CD1c+ conventional DC (P = 0.04) NKT cells (P =0.03) and marked reductions in inflammatory (CD14+CD16+) monocytes (P < 0.0001). Transcriptional profiling of T cell subsets is underway. With 36 patients evaluable (beyond day 100, median follow up of 297 days), the incidence of grade II-IV GVHD is 11.1% in recipients of IL-6R inhibition versus 39.6% in the matched (n = 53) control cohort (P = 0.004). The incidence of grade III/IV acute GVHD is 5.6% in recipients of IL-6R inhibition versus 20.8% in the control cohort (P = 0.045). Protection from grade II-IV acute GVHD was noted in patients receiving both Cy/TBI (7.7% vs. 40.7%, P=0.045) and Flu/Mel conditioning (13.0% vs. 38.5%, P = 0.044). Rates of CMV reactivation were very low in the IL-6R neutralized patients (16.7% vs. 35.8% in controls, P = 0.04), likely due to the prevention of acute GVHD and its' consequent therapy. At one year, the relapse incidence and disease free survival in patients receiving IL-6R inhibition versus the control cohort is 21.2% vs. 30.0% (P = 0.28) and 73.1% vs. 62.4% (P = 0.14) respectively. IL-6 is thus the principal inflammatory cytokine dysregulated after clinical allogeneic SCT and its inhibition appears to offer profound protection from acute GVHD despite robust immune reconstitution, without compromise of the GVL effect. Disclosures:Off Label Use: The use of Tocilizumab to prevent GVHD is experimental and an off label use.

Increased Prevalence of Luminal Narrowing and Stricturing Identified by Enterography in Pediatric Crohn’s Disease Patients With Elevated Granulocyte–Macrophage Colony Stimulating Factor Autoantibodies

Crohn's disease (CD) patients with elevated granulocyte-macrophage colony-stimulating factor autoantibodies (GM-CSF Ab) are more likely to develop stricturing behavior requiring surgery. Computed tomography or magnetic resonance enterography (CTE or MRE) may detect luminal narrowing (LN) before stricture development. The objective of this study was to determine whether CD patients with elevated GM-CSF Ab (≥1.6 μg/mL) have a higher prevalence of LN and stricturing on CTE or MRE. A single-center, cross-sectional study of 153 pediatric patients with CD and control subjects undergoing diagnostic CTE or MRE. Examinations were evaluated for disease activity using a novel scoring system and for the presence of LN, stricture, intra-abdominal abscess, or fistulae. Dichotomous outcomes were compared with respect to antibody status (high or low) using Fisher's exact test and logistic regression, whereas continuous outcomes were evaluated using unpaired t test. GM-CSF Ab were elevated in CD patients (n = 114) with a median (interquartile range) GM-CSF Ab level of 2.3 μg/mL (0.5-6.6 μg/mL) compared with 0.6 μg/mL (0.3-1.3 μg/mL) in healthy and disease control subjects (n = 39) (P = 0.001). Ileal disease location was more common in CD patients with high GM-CSF Ab (P < 0.001). LN increased from 39% in CD patients with low GM-CSF Ab to 71% in those with high levels (P = 0.004) and remained significantly associated with high GM-CSF Ab in a multivariate logistic model, which included age, gender, small bowel location, and duration of disease. Stricturing prevalence on CTE or MRE examination increased from 4% in CD patients with low GM-CSF Ab to 19% in those with high GM-CSF Ab (P = 0.03). Pediatric CD patients with high GM-CSF Ab levels have a higher prevalence of LN on CTE or MRE. Further study will be needed to determine whether medical therapy will reduce progression to stricturing behavior in these patients.

FRI0207 The input of measuring of infliximab and adalimumab levels and levels of antibodies to these drugs in the management of patients with autoimmune diseases treated with anti tnf monoclonal antibodies.

BackgroundIn patients (pts) with RA and other autoimmune diseases the immunogenicity during infliximab (INF) or adalimumab (ADA) therapy lead to the r loss of response. Increasing the drug dose or...

Sublingual immunization with M2-based vaccine induces broad protective immunity (P4301)

Abstract Background: M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n.) route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study we examined whether the sublingual (s.l.) route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored. Methods and Results: A recombinant M2 protein with three tandem copies of the M2e (3M2eC) was expressed in Escherichia coli. Parenteral immunizations of mice with 3M2eC induced high levels of M2e-specific serum Abs but failed to provide complete protection against lethal challenge with influenza virus. In contrast, s.l. immunization with 3M2eC was superior for inducing protection in mice. In the latter animals, protection was associated with specific Ab responses in the lungs. Conclusions: The results demonstrate that s.l. immunization with 3M2eC vaccine induced airway mucosal immune responses along with broad cross-protective immunity to influenza. These findings may contribute to the understanding of the M2-based vaccine approach to control epidemic and pandemic influenza infections.