ELISA Titers Research Articles

Immunogenicity of Non-Living Anthrax Vaccine Candidates in Cattle and Protective Efficacy of Immune Sera in A/J Mouse Model Compared to the Sterne Live Spore Vaccine.

The Sterne live spore vaccine (SLSV, Bacillus anthracis strain 34F2) is the veterinary vaccine of choice against anthrax though contra-indicated for use with antimicrobials. However, the use of non-living anthrax vaccine (NLAV) candidates can overcome the SLSV limitation. In this study, cattle were vaccinated with either of the NLAV (purified recombinant PA (PrPA) or crude rPA (CrPA) and formaldehyde-inactivated spores (FIS of B. anthracis strain 34F2) and emulsigen-D®/alhydrogel® adjuvants) or SLSV. The immunogenicity of the NLAV and SLSV was assessed and the protective efficacies evaluated using a passive immunization mouse model. Polyclonal IgG (including the IgG1 subset) and IgM responses increased significantly across all vaccination groups after the first vaccination. Individual IgG subsets titres peaked significantly with all vaccines used after the second vaccination at week 5 and remained significant at week 12 when compared to week 0. The toxin neutralization (TNA) titres of the NLAV vaccinated cattle groups showed similar trends to those observed with the ELISA titres, except that the former were lower, but still significant, when compared to week 0. The opsonophagocytic assay indicated good antibody opsonizing responses with 75% (PrPA+FIS), 66% (CrPA+FIS) and 80% (SLSV) phagocytosis following spores opsonization. In the passive protection test, A/J mice transfused with purified IgG from cattle vaccinated with PrPA+FIS+Emulsigen-D®/Alhydrogel® and SLSV had 73% and 75% protection from challenge with B. anthracis strain 34F2 spores, respectively, whereas IgG from cattle vaccinated with CrPA+FIS+Emulsigen-D®/Alhydrogel® offered insignificant protection of 20%. There was no difference in protective immune response in cattle vaccinated twice with either the PrPA+FIS or SLSV. Moreover, PrPA+FIS did not show any residual side effects in vaccinated cattle. These results suggest that the immunogenicity and protective efficacy induced by the NLAV (PrPA+FIS) in the cattle and passive mouse protection test, respectively, are comparable to that induced by the standard SLSV.

Pilot Investigation on the Presence of Anti-Hepatitis E Virus (HEV) Antibodies in Piglet Processing Fluids.

Simple SummaryDomestic and wild pigs are the main Hepatitis E virus (HEV) zoonotic reservoirs. Identifying HEV-positive pig farms is important to implement surveillance programs for this emerging zoonotic agent. The aim of this study was to evaluate the use of serosanguineous fluids obtained as part of castration practice (processing fluids (PFs)) to detect anti-HEV antibodies. Ninety-five paired serum and PF samples were collected from newborn piglets of 29 different litters and tested with a commercial ELISA kit. A significant positive correlation (Spearman’s rho: 0.600; p < 0.01) was found between the signal-to-cutoff (S/Co) ratio of anti-HEV antibodies in serum and PF samples. In 26 out of 29 litters (89.7%), there was at least one positive piglet in the serum. Sixteen litters out of 29 (55.2%) were also positive in PFs. The detection of anti-HEV maternal-derived antibodies in PFs confirms a past exposure of sows to the virus. PF may represent a rapid, noninvasive and economical tool to identify HEV-positive farms.Identifying Hepatitis E virus (HEV)-positive pig farms is important to implement surveillance programs for this emerging zoonotic agent. The aim of this study was to evaluate the use of serosanguineous fluids obtained as part of castration practice (processing fluids (PFs)) to detect anti-HEV antibodies in newborn piglets. Ninety-five paired serum and PF samples were collected from piglets of 29 different litters and tested with a commercial ELISA kit. A significant positive correlation (Spearman’s rho: 0.600; p < 0.01) was found between anti-HEV antibodies in serum and PF samples. In 26 out of 29 litters (89.7%), there was at least one positive piglet in the serum. Sixteen litters out of 29 (55.2%) were also positive in PFs. To simulate the use of PF as pooled samples, the limit of detection of the ELISA was assessed mixing the PF sample with strong, medium, medium-weak and weak ELISA titres with 3, 4, 5 and 6 negative PF samples. Our results suggest that it is still possible to identify a positive PF pool when at least one individual PF sample with medium or strong antibody levels is mixed with 5 or 6 individual negative PF samples. The detection of anti-HEV maternal-derived antibodies in PF confirms a past exposure of sows to the virus. PF may represent a rapid, noninvasive and economical tool to identify HEV-positive farms.

Quantification of SARS-CoV-2 antibodies with eight commercially available immunoassays.

Since the emergence of SARS-CoV-2 numerous antibody assays have become available, demonstrating different performance characteristics. This study focused on a quantitative correlation between different commercial assays and a neutralization test (NT). Comparative data is needed as a basis for the production of convalescent plasma and potential interpretations COVID-19 immunity.Sera of 100 SARS-CoV-2 convalescent plasma donors were collected and SARS-CoV-2 antibodies were characterized using three different IgG-ELISAs (EUROIMMUN IgG and NCP-IgG ELISA, Wantai ELISA), two CLIA (Elecsys, LIAISON) and two lateral flow tests (MEDsan IgM/IgG-Rapid-Test, Wantai Rapid Test) and subsequently correlated to neutralization titers. The Wantai ELISA and the Elecsys provide the highest sensitivities in this sample (98 and 95 percent respectively). Titers with the best overall quantitative correlation to the NT titer were obtained with the Euroimmun IgG ELISA assay (Rho=0.759) and the Wantai ELISA assay (Rho=0.729).An infection without fever and negative or weakly positive reactions in the Wantai Rapid test were negative predictive factors for NT titers >1:200 (negative predictive value of 92 % and 92 % respectively, combination of both 100 %).The Wantai ELISA titer could be a suitable substitute for NT. An adequate pooling strategy of plasma units additionally could compensate deviations of individual antibody titers.

Supplemental dietary selenium enhances immune responses conferred by a vaccine against low pathogenicity avian influenza virus

Selenium is a trace mineral that has antioxidant activities and can influence the immune system. However, antiviral effects of selenium have not been well studies in chickens. Chickens were therefore fed diets supplemented with two levels of two different sources of selenium (organic: selenium enriched yeast; SEY or inorganic: sodium selenite; SS). Chickens in the control groups did not receive supplemental dietary selenium. At 14 and 21 days of age, chickens were vaccinated with an inactivated low pathogenicity avian influenza virus (AIV, subtype H9N2) vaccine and blood samples were collected to determine the level of antibodies using hemagglutination inhibition (HI) and ELISA. At 30 days of age, chickens were also challenged with the same virus and swab samples were collected to assess the amount of virus shedding. Antibody levels, as measured by HI, increased significantly in the chickens that received higher levels of SEY at 16 days post vaccination. ELISA titers for IgM and IgY were higher in selenium supplemented chickens. Comparing to challenged control, virus shedding was lower in organic as well as inorganic selenium treated groups. Therefore, it may be concluded that supplemental dietary selenium could enhance vaccine conferred immunity thereby impacting protection against viral challenge in chickens.

Development and use of indirect liquid-phase ELISA test system for detection of PRRS virus antigen during in-process control of raw materials intended for vaccine production

Porcine reproductive and respiratory syndrome (PRRS) being endemic and reported in the most countries in the world remains one of the most challenging diseases in pig industry. The main disease control measures include preventive vaccination and animal movement control within and outside the country as well as diagnostic testing of pigs in the population. Live and inactivated vaccines are used for specific prevention of porcine reproductive and respiratory syndrome. Complete and irreversible infectious agent inactivation with maximum epitope preservation and protective immunity in immunized animals are the main requirements for inactivated vaccines. Therefore, continuous improvement of methods for vaccine quality control at various vaccine production stages is of current importance. Results of development of the test system based on indirect liquid-phase enzyme-linked immunosorbent assay (ELISA) for PRRS virus antigen detection and activity testing in infectious and inactivated virus-containing cell cultures at intermediate stages of the vaccine production process are described in the paper. The test-system development process included purified and concentrated virus antigen as well as hyperimmune rabbit sera preparation. Specificity of purified and concentrated virus antigen was confirmed with real-time polymerase chain reaction. The developed test-system was shown to detect the virus antigen at initial infectivity titre of 4.87–7.21 lg TCID50/cm3 corresponding to ELISA titre (dilution) of 1:4 up to 1:64. Methodical Guidelines for detection of porcine reproductive and respiratory syndrome virus antigen with indirect liquid-phase enzyme-linked immunosorbent assay (ELISA) (2019) were developed based on the work results, commissioned and approved by the FGBI “ARRIAH” Scientific Board.

Immunostimulant effect of a mixed herbal extract on infectious bursal disease virus (IBDV) vaccinated chickens in the context of a co-infection model of avian influenza virus H9N2 and IBDV

This study was conducted to assess the comparative effects of a mixed herbal extract (MHE) containing Ocimum sanctum, Withania somnifera, Emblica officinalis, Tinospora cordifolia, Mangifera indica, and Asphaltum (shilajit) on infectious bursal disease virus (IBDV)-vaccinated (VAC) chickens infected with IBDV and avian influenza virus (AIV) H9N2. The experiment included three groups (G1-G3): G1, the negative control group; G2, the VAC + challenged (Ch) group; and G3, the VAC + Ch + MHE group. MHE was orally administered continuously for 5 weeks post-vaccination (PV) with IBDV at 12 days of age, and the chicks were simultaneously challenged with virulent IBDV (intraocularly) and AIV H9N2 (intranasally) at 21 days PV. Blood and tissue samples as well as tracheal and cloacal swabs were gathered at different times PV and post-challenge. Immunological and haematological parameters, histopathological lesions, relative organ weights and final live weights revealed significant differences (P ≤ 0.05) between G2 and G3 groups. Furthermore, in the G3 group, the protection rates, ELISA and HI titers and CD4+/CD8+ ratio were significantly increased, whereas viral shedding titers and the heterophil/lymphocyte ratio were decreased. In conclusion, the oral administration of the mixed herbal extract for 5 weeks can stimulate the immune response to IBDV vaccination and relieves the pathogenicity of an AIV H9N2 and IBDV co-infection in chickens.

Macrophage production and activity of innate immune proteins C1q, C1r, and C1s are modulated in response to molecular patterns

Abstract Pattern recognition receptors of the complement system, such as C1q, are vital for clearance of foreign and altered-self targets. C1q has a dual role in vivo: activating the inflammatory classical complement pathway via C1 complex formation (C1qC1r2C1s2) and opsonization in the absence of C1r and C1s which is anti-inflammatory. We hypothesize that macrophages encountering foreign targets will increase levels of C1 subunits to activate C1 and mount an inflammatory response while macrophages encountering damaged-self targets may only increase C1q production to promote anti-inflammatory responses and clearance. Human monocyte derived macrophages (HMDM) were incubated with LPS, E. coli bioparticles, oxLDL, apoptotic cells or polarized into M1 or M2 HMDMs. RNA was isolated for qPCR and supernatant for Luminex protein analysis, ELISA, and C1(q) hemolytic titers. Data show that M1 HMDM increase C1r and C1s mRNA, and produce higher levels of active C1 than M0 HMDM. In contrast, there is no change in gene or protein expression of C1 subunits in M2 HMDM. HMDM treatment with foreign targets revealed a trend of increased C1r and C1s mRNA, and decreased C1q mRNA, but not at the protein level. Treatments with damaged-self targets varied; as hypothesized, C1q gene and protein expression increases in response to apoptotic cells, however there are no substantial changes in treatments with oxLDL. Significant levels of active C1q, but not C1, were detected in response to LPS, oxLDL, and apoptotic cells. Data suggests there is modulation of C1 subunit production and activity in response to different targets. C1q production in the absence of C1r and C1s may be important in resolving inflammation during clearance of apoptotic cells, thus avoiding autoimmunity.

Characterization of serological response to dengue and Zika viruses in pregnant women during the Zika outbreak in Brazil

Abstract The outbreak of Zika virus (ZIKV) and associated congenital Zika syndrome in dengue virus (DENV)-endemic regions raised questions about the effects of ZIKV infection on DENV immunity and disease outcome and vice versa, which highlighted the need of serological tests that can discriminate between DENV and ZIKV infections. Previously we developed combined DENV and ZIKV non-structural protein 1 (NS1) enzyme-linked immunosorbent assays (ELISAs) to overcome the cross-reactivity of traditional envelope protein-based serological tests (Tsai et al. Clin Infect Dis 2017;65:1829). In this study, we used this assay to screen 120 serum samples collected from asymptomatic parturient women during the ZIKV outbreak in Salvador, Brazil and identified four serostatus groups. We further characterized antibody responses in 8 participants including 2 primary DENV (pDENV), 2 secondary DENV (sDENV), 1 primary ZIKV (pZIKV), and 3 ZIKV with previous DENV (ZIKVwprDENV) infections. Using depletion experiments with inactivated ZIKV and urea tests, we determined the IgG avidity to DENV. Using depletion experiments with inactivated DENV and endpoint ELISA titers, we determined ZIKV type-specific and cross-reactive antibody and found pZIKV had higher proportion of ZIKV type-specific antibody (82%) than ZIKVwprDENV (10-16%). These results were supported by microneutralization tests. Our findings suggest that combined DENV and ZIKV NS1 IgG ELISAs plus depletion experiments can delineate past and present DENV and ZIKV infections and are a potential tool to further our understanding of pathogenesis and epidemiology of DENV and ZIKV in endemic regions. Research supported by R01 AI11076901, R21AI135292-01A1 from NIAID, and P30GM114737 from NIGMS, NIH.

Treatment of 5 Critically Ill Patients With COVID-19 With Convalescent Plasma

Coronavirus disease 2019 (COVID-19) is a pandemic with no specific therapeutic agents and substantial mortality. It is critical to find new treatments. To determine whether convalescent plasma transfusion may be beneficial in the treatment of critically ill patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Case series of 5 critically ill patients with laboratory-confirmed COVID-19 and acute respiratory distress syndrome (ARDS) who met the following criteria: severe pneumonia with rapid progression and continuously high viral load despite antiviral treatment; Pao2/Fio2 <300; and mechanical ventilation. All 5 were treated with convalescent plasma transfusion. The study was conducted at the infectious disease department, Shenzhen Third People's Hospital in Shenzhen, China, from January 20, 2020, to March 25, 2020; final date of follow-up was March 25, 2020. Clinical outcomes were compared before and after convalescent plasma transfusion. Patients received transfusion with convalescent plasma with a SARS-CoV-2-specific antibody (IgG) binding titer greater than 1:1000 (end point dilution titer, by enzyme-linked immunosorbent assay [ELISA]) and a neutralization titer greater than 40 (end point dilution titer) that had been obtained from 5 patients who recovered from COVID-19. Convalescent plasma was administered between 10 and 22 days after admission. Changes of body temperature, Sequential Organ Failure Assessment (SOFA) score (range 0-24, with higher scores indicating more severe illness), Pao2/Fio2, viral load, serum antibody titer, routine blood biochemical index, ARDS, and ventilatory and extracorporeal membrane oxygenation (ECMO) supports before and after convalescent plasma transfusion. All 5 patients (age range, 36-65 years; 2 women) were receiving mechanical ventilation at the time of treatment and all had received antiviral agents and methylprednisolone. Following plasma transfusion, body temperature normalized within 3 days in 4 of 5 patients, the SOFA score decreased, and Pao2/Fio2 increased within 12 days (range, 172-276 before and 284-366 after). Viral loads also decreased and became negative within 12 days after the transfusion, and SARS-CoV-2-specific ELISA and neutralizing antibody titers increased following the transfusion (range, 40-60 before and 80-320 on day 7). ARDS resolved in 4 patients at 12 days after transfusion, and 3 patients were weaned from mechanical ventilation within 2 weeks of treatment. Of the 5 patients, 3 have been discharged from the hospital (length of stay: 53, 51, and 55 days), and 2 are in stable condition at 37 days after transfusion. In this preliminary uncontrolled case series of 5 critically ill patients with COVID-19 and ARDS, administration of convalescent plasma containing neutralizing antibody was followed by improvement in their clinical status. The limited sample size and study design preclude a definitive statement about the potential effectiveness of this treatment, and these observations require evaluation in clinical trials.

Growth and humoral immune effects of dietary Original XPC in layer pullets challenged with Mycoplasma gallisepticum

Effects of dietary Original XPC (XPC) in commercial layer pullets challenged with the virulent, low passage R strain of Mycoplasma gallisepticum (Rlow MG) were investigated. Hy-Line W-36 pullets sourced from MG-clean breeders were fed a basal diet with or without (CON) XPC (1.25 kg/metric ton) from hatch until 12 wk of age (woa). At 8 and 10 woa, half of the birds in each dietary treatment were challenged with Rlow MG. Body weight was recorded at 3, 8, and 12 woa, and ovary, ceca, and bursa weights were recorded at 3 and 12 woa. Blood samples were taken immediately before the initial Rlow MG challenge at 8 woa and again at 12 woa to test for IgM and IgG antibody production against MG. All birds were evaluated for MG lesion scores at 12 woa. Regardless of challenge, inclusion of XPC in the diet did not significantly alter BW at 3 or 8 woa or relative organ weights at 3 or 12 woa. However, at 12 woa, BW of XPC-fed birds, regardless of challenge was significantly (P = 0.0038) heavier than CON by 25.7 g. All birds tested negative for MG antibodies before the 8 woa challenge. Respective percentage serum plate agglutination and ELISA positive birds at 12 woa were 0 and 0% (CON, nonchallenged), 1.4 and 0% (XPC, nonchallenged), 100 and 47.2% (CON, challenged), and 100 and 50.0% (XPC, challenged). Diet did not significantly affect ELISA titers, but they were significantly (P < 0.0001) increased due to challenge. Furthermore, lesion scores were significantly higher for Rlow MG-challenged birds (P = 0.0012), and dietary treatment with XPC in challenged birds numerically reduced MG lesion scores from 0.278 to 0.194. In conclusion, although dietary XPC did not significantly alter the humoral immune response, antibody titer levels, or severity of MG lesions in layer pullets that were or were not challenged with Rlow MG, it led to an increase in their rate of growth through 12 woa.

Immunogenicity and Efficacy of Live Infectious Bronchitis 793/B.08IR Vaccine in SPF Chickens

Infectious bronchitis virus (IBV) has a variety of serotypes with relatively limited cross-protection leading the disease to be a major problem in the poultry industry. The IBV 793/B strain has identified to circulate in Iran; therefore, the development of a specific vaccine to protect against the virulent virus has received attention. In this regard, the live IB 793/B vaccine (793/B.08IR) was developed in the Razi Vaccine and Serum Research Institute. In this study, the immunogenicity of 793/B.08IR vaccine via different routes of vaccination and efficacy of the vaccine were determined in specific-pathogen-free (SPF) chickens. Three treatment groups of 10 SPF chickens received the vaccine via eye drops, spray, and drinking water. The sera were collected from the chicks at 3 and 6 weeks after the vaccination, and IBV specific antibody was measured using enzyme-linked immunosorbent assay (ELISA) and serum neutralization (SN) test. To evaluate 793/B.08IR vaccine efficacy, 10 SPF chickens were vaccinated using eye drops. Moreover, 10 unvaccinated chickens were separately retained as negative controls. The birds were challenged with the virulent virus 3 weeks following the vaccination. Five days after the challenge, the tracheal swab was taken for virus reisolation. In the immunogenicity test, the ELISA titers of three vaccinated groups were significantly higher than the background values obtained in the control group (p&amp;lt;0.0001). The mean value of ELISA titer in the spray vaccinated group was higher than the spray and drinking water vaccinated groups 3 weeks following the vaccination; however, the difference was not statistically significant. No differences were observed in antibody titers among the three vaccinated groups 6 weeks after the vaccination. The results of the SN test confirmed the data obtained from the ELISA. The results of antibody titer and its increasing trend in chickens showed that 793/B.08IR vaccine induce proper immunity against the virus. In the efficacy test, IBV was isolated from 90% of the unvaccinated controls and 10% of vaccinated groups. The results of the recovery of the virus after the challenge showed that 793/B.08IR vaccine can provide a significantly improved protection against the pathogen in SPF vaccinated chickens.

The use of polyazolidineammonium and dimethyl-sulfoxide antigen Yersinia pseudotuberculosis to obtain hyperimmune serum

The use of polyazolidinammonium modified with iodine hydrate ions (PAAG) as an adjuvant made it possible to obtain rabbit hyperimmune blood serums for dimethyl-sulfoxide antigen (DA) of a pseudotuberculosis microbe with generic specificity. Antibody titers in ELISA with Y. pseudotuberculosis and Y. enterocolitica cells amounted to 1:25600-1:12800, and with cells of other genera of intestinal microflora 1:100-1:400. The optimal immunizing dose for obtaining hyperimmune yersiniosis serum was a dose of 2 mg DA of Y. pseudotuberculosis per rabbit. Such a dose made it possible to obtain hyperimmune sera with a high titer of specific antibodies with a small consumption of antigen. The optimal concentration of PAAG solution for hyperimmunization of Y. pseudotuberculosis DA rabbits was 1%.

Camel hydatidosis diagnostic kit: optimization of turnip and horseradish peroxidase conjugates using glutaraldehyde method.

Echinococcosis/hydatidosis is one of the most important parasitic zoonotic diseases in the world. Cystic echinococcosis increases public health and socio-economic concern due to considerable morbidity rates that give rise to elevated economic losses both in the public health part and in the farm animal field. The enzyme linked immunosorbent assay (ELISA) is consider the more accurate tool for diagnosis of hydatidosis in camels. In the present study, affinity purified Echinococcus granulosus (E. granulosus) antigens (APA) were purified from crude hydatide E. granulosus germinal layer proteins for detection of E. granulosus antibodies in infected camels, using affinity matrix (camel IgGs coupled to CNBr-activated Sepharose). The electrophoretic profile of the APA showed that it was separated into two bands; one major band of 130kDa and one minor band at 55kDa. These antigens were used successfully as specific coating antigenic proteins in detection of echinococcosis in camel. In a trial to prepare an anti-camel IgGs peroxidase conjugate; peroxidase enzyme was purified from turnip roots (TPOD) using ammonium sulfate precipitation and affinity chromatography on phenyl Sepharose CL-4B. The purified TPOD showed a major band at 35kDa. Rabbit anti-camel IgG antibodies (AC IgGs) were prepared then purified using affinity chromatography on Protein G-Sepharose. The TPOD, and commercial HRP for comparison, enzymes were conjugated to AC IgGs using 1%, 5% and 10% glutaraldehyde. The results revealed that the HRP was much better than TPOD in conjugation with AC-IgG antibodies and the 10% glutaraldehyde concentration was the most efficient concentration with ELISA titer 1:50.

Early antibody response and clinical outcome in experimental canine leishmaniasis

Infected dogs are the main reservoir of zoonotic visceral leishmaniasis, a widespread parasitic disease caused by Leishmania infantum. Therefore, the control of canine infections is required to reduce the incidence of human cases. Disease outcome in dogs depends on the fine balance between parasite virulence and efficacy of the immune system. Thus, knowledge of early response could yield relevant information for diagnosis and follow-up. In our study, 20 Beagle dogs were intravenously infected with 108 amastigotes of a fresh isolate of L. infantum and monitored along 16 weeks post inoculation. Specific antibody response and clinical evolution of infected animals were highly variable. Immunofluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA) were useful to assess infection status, although only ELISA with promastigote-coated plates and, particularly, western blotting (WB) allowed an early diagnosis. Prominent antigens were identified by mass peptide fingerprinting. Chaperonin HSP60, 32 and 30 KDa antigens were recognized by all dogs on week 10 post infection. This suggests that these antigens may be valuable for early diagnosis. Advanced infection showed, in addition, reactivity to HSP83 and HSP70. Disease outcome did not show a clear relationship with ELISA or IFAT titers. Correlation between the clinical status and the combined reactivity to some antigens sustains their use for diagnosis and follow-up.

Epitope Dependent Augmentation of the Immune Response in Hemophilia Α Mice Immunized with Factor VIII/Antibody Immune Complexes

Introduction: The immune response to factor VIII (fVIII) is a CD4+ T cell dependent process initiated by fVIII recognition and processing by antigen presenting cells. The C1 and C2 domains have been proposed as the primary domains that mediate fVIII internalization by dendritic cells. Our prior studies demonstrate that fVIII pre-bound to anti-C1 and C2 domain monoclonal antibodies (MAbs) reduces fVIII internalization by murine derived dendritic cells. However, anti-A1 and A3 domain antibodies increase fVIII endocytosis by dendritic cells. In this study, we analyzed the antibody titers of hemophilia A mice immunized with binary fVIII and MAb complexes to determine whether changes in fVIII internalization observed in vitro correspond to changes in the immune response to fVIII in vivo. Methods: Exon16 (E16) knockout mice deficient in fVIII were immunized with B-domain deleted fVIII in the presence of either anti-fVIII MAb 2-116 (anti-A1, IgG2a), 4A4 (anti-A2, IgG2a), 2-113 (anti-A3, IgG1), B136 (anti-C1, IgG2a), or 3D12 (anti-C2, IgG2b) versus fVIII alone. Mice were immunized by weekly retro-orbital injections of 0.1 µg fVIII incubated with 1 µg anti-fVIII MAb for 4 weeks followed by a boost dose of 0.2 µg fVIII and 2 µg anti-fVIII MAb one week later at week 5. Anti-fVIII ELISA titers (in arbitrary units, AU) were analyzed from plasma samples collected at week 7 to account for IgG half-life. A separate cohort of mice immunized with anti-fVIII MAbs alone served as controls to normalize ELISA titers in each fVIII/MAb group to account for residual injected MAbs in the plasma samples. Results: Hemophilia A mice immunized with fVIII/2-116 and fVIII/4A4 complexes significantly increased anti-fVIII ELISA titers compared to mice immunized with fVIII alone (figure 1). Mice immunized with fVIII/2-116 and fVIII/4A4 had median ELISA titers of 7,401 AU (interquartile range, IQR, 2,654 - 11,910 AU) and 3,620 AU (IQR 1,062 - 7,969 AU), respectively, compared to mice immunized with fVIII (median titer 1,063 AU, IQR 402 - 2,476 AU). MAb 2-116 is a non-inhibitory antibody with a titer of &lt;1 Bethesda Unit (BU)/mg IgG, while 4A4 is a highly inhibitory antibody with a titer of 40,000 BU/mg IgG. Neither MAb interferes with fVIII binding to von Willebrand factor (VWF) or phospholipid vesicles. Mice immunized with fVIII/2-113 (median titer 2,210 AU, IQR 402 - 8,318 AU), fVIII/B136 (median titer 123 AU, IQR 0 - 9,709 AU), and fVIII/3D12 (median titer 3,244 AU, IQR 0 - 5,180 AU) did not have significantly different anti-fVIII ELISA titers compared to mice immunized with fVIII. However there was a trend towards reduced anti-fVIII titers with fVIII/B136 and fVIII/3D12 injections. MAbs B136 and 3D12 inhibit fVIII binding to VWF and have been shown to significantly increased fVIII clearance in hemophilia A mice compared to fVIII in a VWF-dependent manner. Median titers for mice immunized with MAbs alone to account for residual injected MAbs were 13 AU (2-116), 12 AU (4A4), 18 AU (2-113), 16 AU (B136), and 4 AU (3D12). Conclusions: Immunization of hemophilia A mice with fVIII/MAb complexes, specifically anti-A1 MAb 2-116 and anti-A2 MAb 4A4, enhance the immune response to fVIII. MAb 2-116 significantly increased anti-fVIII antibody titers in vivo, which correlates with increased fVIII internalization by immature dendritic cells observed in vitro. A better understanding of the effect of anti-fVIII antibodies on fVIII conformational changes could provide insight into whether these changes alter fVIII recognition by immune cells and subsequently propagate the immune response to fVIII at the onset of inhibitor formation or during immune tolerance induction. Disclosures Batsuli: Genentech: Other: Advisory board participant; Bayer: Other: Advisory board participant; Octapharma: Other: Advisory board participant. Meeks:HEMA Biologics: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Takeda-Shire: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees.

The Immune Response to Murine Factor VIII in Single Exon Deletion and Total Gene Deletion Murine Models of Hemophilia a

Introduction: Though a vital therapy for patients with hemophilia A, exposure to factor VIII (fVIII) can lead to the development of neutralizing anti-fVIII IgG. Interestingly, not all patients with hemophilia A form inhibitors, with approximately 20-30% of patients with severe hemophilia A and 5% of patients with mild to moderate hemophilia A developing inhibitors. Several factors have been hypothesized to govern the propensity of patients to develop inhibitors, including the type of F8 gene mutation a patient possesses. Approximately, 60-70% of hemophilia A patients with large deletions (greater than 50 base pairs) of the F8 gene form inhibitors, while only 20-30% of patients with smaller deletions develop inhibitors. However, no significant difference in inhibitor formation has been observed between single exon 16 (E16) and complete (TKO) F8 gene deficient hemophilia A mice exposed to human fVIII (hfVIII). As E16 mice are on a mixed S129-B6 background and strain differences can impact the immunological outcome to a given immunogen, it is possible that the mixed background influenced the humoral immune response to hfVIII in E16 mice. However, E16 mice that are backcrossed &gt;10 generations onto the same B6 background as TKO mice (B6-E16) were found to develop a comparable IgG response to hfVIII as B6-TKO and S129/B6-E16 mice. As human and murine B domain deleted (BDD) fVIII are 85% identical, we hypothesize that the diversity between human and murine fVIII may not allow for elucidation of immune differences that may exist between hemophilia A mice with large or small F8 mutations. Accordingly, we herein sought to compare the relative immunogenicity of murine fVIII (mfVIII) in these distinct hemophilia A mice. Methods:S129/B6 E16, B6-E16 and B6-TKO mice were administered four weekly retro-orbital infusions of 1 μg recombinant BDD mfVIII or hfVIII. Two weeks later, mice were challenged with a 2 μg dose of recombinant mfVIII or hfVIII. One week prior to challenge and one-week post challenge, plasma was collected for examination of anti-fVIII IgG titers by an enzyme linked immunosorbent assay. Results:Consistent with previous reports, no statistically significant difference in anti-hfVIII IgG titers were observed between S129/B6-E16, B6-E16 and B6-TKO mice. When exposed to mfVIII the majority of B6-TKO mice developed inhibitors (87.5%), while the single exon B6-E16 or S129/B6-E16 mice had 50% and 56% rates of inhibitor development, respectively. The B6-E16 mice had a lower median anti-mfVIII IgG ELISA titer of 12.3 (range 1 to 190.3) compared to S129/B6-E16 and B6-TKO that had median ELISA titers of 473 and 289, respectively. When assessing the anti-mfVIII IgG titers only in the mice that had detectable titer levels, the IgG titers for S129/B6-E16 mice ranged from 34.2 to 1519.4 and for B6-TKO mice ranged from 31.3 to 1377. Conclusion: Given the difference in rates of immune response to mfVIII in mice carrying a single exon or total F8 gene deletion, mfVIII may be useful to elucidate the correlation between F8 gene mutations and the propensity of an individual to respond to factor replacement therapy, and in particular modulators of the immune response in lower risk F8 gene mutations. Disclosures Batsuli: Genentech: Other: Advisory board participant; Octapharma: Other: Advisory board participant; Bayer: Other: Advisory board participant. Meeks:HEMA Biologics: Membership on an entity's Board of Directors or advisory committees; Takeda-Shire: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees.

2712. Safety and Immunogenicity of two Doses of ExPEC4V Vaccine Against Extraintestinal Pathogenic Escherichia coli Disease in Healthy Adult Participants

BackgroundThe ExPEC4V vaccine contains 4 Escherichia coli O-antigens (O1A, O2, O6A, O25B) conjugated to exotoxin protein A and is being studied for prevention of Invasive Extraintestinal pathogenic E. coli (ExPEC) Disease (IED). This phase-2 double-blind study assessed safety and immunogenicity of ExPEC4V Clinical Trial Material (CTM), manufactured via a redesigned process (optimized O1A strain).MethodsParticipants (≥18 years) in stable health were randomized (3:1) to receive ExPEC4V dose 4:4:4:8 μg PS/serotype or placebo on Day 1 and second vaccination on Day 181 (6 months after first vaccination). Participants will be followed for safety until end of study at Day 360. Reactogenicity and immunogenicity (by ELISA, opsonophagocytic killing [OPA] assays) were evaluated pre-vaccination, and 15 days after first and second vaccinations (Day 195).ResultsOf 100 participants randomized (mean age 56, 48% males) and vaccinated (ExPEC4V, n = 75; placebo, n = 25), 97 completed Day 30. Solicited local AEs were higher for ExPEC4V (38.7%) than placebo (20%); most frequent was pain/tenderness (38.7% vs 20%). Solicited systemic AEs were higher in ExPEC4V (49.3%) than placebo (20%); most frequent was fatigue (32% vs. 12%). No serious or grade 3 solicited local AEs were reported. One participant in ExPEC4V experienced a grade 3 solicited systemic fatigue considered vaccine-related by investigator. ExPEC4V demonstrated immune responses against all serotypes at Day 15. Geometric mean titer effective concentration rank by serotypes was O2 > O1A > O6 > O25B (Figures 1 and 2). At Day 15, ≥ 82% of participants in ExPEC4V and none in placebo had ≥2-fold increase from baseline of ELISA titer for all serotypes. In ExPEC4V, ≥47% had ≥2-fold increase from baseline of OPA titer for all serotypes, while 8% in placebo had ≥2-fold increase only for O6A. Good correlation was observed between ELISA and OPA across serotypes (r ≥ 0.76).ConclusionExPEC4V elicited robust and functional immune responses across all serotypes and was well tolerated with no vaccine safety findings. This study supports the development of future multivalent ExPEC vaccine to prevent IED. Disclosures All authors: No reported disclosures.

Amyloid and Tau in Neurodegeneration

Background and Hypothesis:Familial British dementia (FBD) and familial Danish dementia (FDD) are two autosomal dominant neurodegenerative diseases caused by mutations in the BRI2 gene. FBD and FDD are characterized by widespread cerebral amyloid angiopathy (CAA), parenchymal amyloid deposition, and neurofibrillary tangles.Previous studies have shown that accumulation of Danish amyloid peptide (ADan) induces hyperphosphorylation of tau and cell cytotoxicity; however, progress in our understanding of the interaction between ADan and tau has been hindered by the lack of antibodies against ADan. In addition, whether the British amyloid peptide (ABri) has a similar effect on tau phosphorylation remains unknown. The main goals of our project are to generate monoclonal antibodies against ADan and to test whether the ABri peptide induces hyperphosphorylation of tau and cell cytotoxicity.&#x0D; Experimental Design or Project Methods:A peptide homologous to the C-terminus of the ADan peptide was used to immunize 5 mice. Serum samples were tested from mice with high ELISA titers and the best 2 animals were used for cell fusion. Cell culture supernatants were screened by ELISA, western blot and immunohistochemistry. ABri peptides were used as negative controls.HEK cells expressing human tau were used to assess the interaction between ABri and tau. We performed immunocytochemistry and Western blot analysis to investigate tau phosphorylation.&#x0D; Results:A total of 11 clones tested positive by western blot. 3 clones tested positive for ABri and were discarded. 5 clones were tested by immunohistochemistry. 3 of the positive clones will be used for subcloning to complete clonality. Work in progress on the interaction between ABri and tau will determine whether ABri induces tau hyperphosphorylation in cell culture.&#x0D; Conclusion and Potential Impact:Our work will provide novel tools to study the interaction between amyloid and tau in neurodegenerative diseases and may uncover possible new targets for pharmaceutical intervention in tauopathies.

Scalable, semi-automated fluorescence reduction neutralization assay for qualitative assessment of Ebola virus-neutralizing antibodies in human clinical samples.

Antibody titers against a viral pathogen are typically measured using an antigen binding assay, such as an enzyme-linked immunosorbent assay (ELISA), which only measures the ability of antibodies to identify a viral antigen of interest. Neutralization assays measure the presence of virus-neutralizing antibodies in a sample. Traditional neutralization assays, such as the plaque reduction neutralization test (PRNT), are often difficult to use on a large scale due to being both labor and resource intensive. Here we describe an Ebola virus fluorescence reduction neutralization assay (FRNA), which tests for neutralizing antibodies, that requires only a small volume of sample in a 96-well format and is easy to automate. The readout of the FRNA is the percentage of Ebola virus-infected cells measured with an optical reader or overall chemiluminescence that can be generated by multiple reading platforms. Using blinded human clinical samples (EVD survivors or contacts) obtained in Liberia during the 2013–2016 Ebola virus disease outbreak, we demonstrate there was a high degree of agreement between the FRNA-measured antibody titers and the Filovirus Animal Non-clinical Group (FANG) ELISA titers with the FRNA providing information on the neutralizing capabilities of the antibodies.

Antibody Response to Feline Calicivirus Vaccination in Healthy Adult Cats.

This study evaluated the prevalence of feline calicivirus (FCV) antibodies and response to vaccination in healthy adult cats. Cats >1 year (n = 111) that had not been vaccinated within 12 months of enrollment in the study received a vaccine containing inactivated FCV antigen strains 431 and G1. Antibodies were determined on Days 0, 7, and 28 by virus neutralization (VN) using FCV isolate KS20, and by broad spectrum blocking FCV enzyme-linked immunosorbent assay (ELISA). Factors associated with the presence of antibodies and vaccine response were determined by uni- and multivariate analysis. Pre-vaccination antibodies were detected in 62.2% of cats (CI95%: 52.9–70.1) by VN and in 77.2% (CI95%: 67.5–84.6) by ELISA. A ≥4-fold titer increase after vaccination was observed in 13.6% (CI95%: 8.3–21.4) of cats with VN and 33.7% (CI95%: 24.5–44.5) with ELISA. Factors associated with the presence of pre-vaccination VN antibodies were age (≥2 years; OR: 7.091; p = 0.022) and lack of previous vaccination (OR: 3.472; p = 0.014). The presence of pre-vaccination ELISA antibodies was associated with time since last vaccination (OR: 5.672; p = 0.043). Outdoor cats were more likely to have a ≥4-fold ELISA titer increase (OR: 5.556; p = 0.005). Many cats had pre-vaccination FCV antibodies, and their presence depended on previous vaccinations and increases with age. A ≥4-fold titer increase was rarely observed and was influenced by the lifestyle of the cat.