ELISA Titers Research Articles

P-1159. S. aureus α-toxin Neutralization Antibody Reduces Risk for Recurrent Skin and Soft Tissue Infection in Children

Abstract Background Staphylococcus aureus is the leading bacterial cause of infectious-related mortality worldwide. To date, immune correlates of protection are undefined, resulting in unsuccessful vaccine development attempts. Anti-S. aureus α-toxin (Hla) IgG response is the only identified correlate of protection against recurrent infection in children. We aimed to correlate the anti-Hla neutralization antibody (Nab) response against risk of recurrent S. aureus infection. Methods We utilized serum and plasma samples from two prospective, observational studies enrolling children: 1) healthy controls with or without S. aureus colonization, 2) culture-confirmed S. aureus skin and soft tissue infection (SSTI), or 3) acute invasive S. aureus infection. Patients with risk factors for healthcare-associated S. aureus infections were excluded. Acute blood samples were drawn at enrollment/acute infection and convalescent samples 4-8 weeks later. Fold induction titer was calculated as a ratio between the convalescent and acute anti-Hla Nab titer. All participants were followed longitudinally every 3 months for 1 year to document recurrent infection. Anti-Hla IgG was determined by ELISA and anti-Hla Nab titer by RBC lysis assay. Results A total of 254 participants were included in the overall analyses. Children with SSTI had a 4.77 (95% CI: 2.15 to 10.19) increased odds of having a recurrent infection and were younger (mean age: 2.51 vs 5.17 years, p< 0.0001) compared to those with invasive infection (Table 1). Acute and convalescent titers were compared across patients: children with invasive infections had the highest acute anti-Hla NAb titers while non-colonized controls had the lowest (1.62 vs. 0.55, p< 0.0001). Children with SSTI and no recurrent infections had higher convalescent to acute anti-Hla NAb titers. Among patients with SSTI, for each unit increase in the log fold induction anti-Hla NAb titer, children with SSTI had a 0.54 (95% CI: 0.31-0.96) decreased odds of recurrent infection within 12 months of follow-up. We did not see a similar trend in the invasive group. Conclusion Higher anti-Hla NAb fold induction titer correlated significantly with a decreased risk of recurrent SSTI infection. Our study supports the advancement of Hla as an antigenic target for S. aureus vaccine development. Disclosures Kristina G. Hulten, PhD, Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support Jonathon C. McNeil, MD, Nabriva: site investigator on clinical trial Sheldon L. Kaplan, MD, Pfizer: Grant/Research Support Juliane Bubeck-Wardenburg, MD, PhD, Aridis Pharmaceuticals: I have a financial agreement with Aridis Pharmaceuticals related to patents owned by the University of Chicago|Forward Defense, LLC: I may receive royalty income based on a technology developed in my laboratory that is currently owned by Washington University and subject to licens|Forward Defense, LLC: Ownership Interest

The Role of Complement Activation in IgM M-Protein-Associated Neuropathies.

Polyneuropathy associated with an immunoglobulin M (IgM) monoclonal gammopathy is characterized by slowly progressive, predominantly distal sensorimotor deficits, sensory ataxia, and electrophysiologic features of demyelination. IgM antibodies against myelin-associated glycoprotein (MAG) are present in serum from most patients. Nerve damage most likely results from the concerted action of binding of anti-MAG antibodies to nerves, followed by complement activation. The interaction of anti-MAG antibodies with complement activation and their relation to clinical characteristics have not been studied in detail. We studied the correlation among anti-MAG antibody titers, complement activation, and IgM-associated polyneuropathy disease severity. We used serum samples from 101 patients with IgM-associated polyneuropathy to assess IgM anti-MAG titers by ELISA and antibody-mediated complement deposition using both an ELISA-based system and a cell-based system of primate peripheral nerve slides. We studied correlations of complement activation with anti-MAG ELISA titers and clinical characteristics. IgM anti-MAG titers varied from negative to strongly positive. Complement deposition in the ELISA-based system correlated significantly with anti-MAG antibody titer (Spearman rho 0.80; p < 0.0001) despite large variability between serum samples with comparable anti-MAG titers. This variability was even larger in the cell-based assay, which also showed complement deposition in IgM anti-MAG negative patients, indicating the presence of autoantibodies directed against epitopes other than MAG in a subset of patients with IgM-associated polyneuropathy. Clinical characteristics did not correlate with anti-MAG titers or complement activation. Anti-MAG antibody titers correlate with the level of complement activation in both ELISA and cell-based systems. However, clinical characteristics of IgM-associated polyneuropathy do not or only weakly correlate with titers or the level of complement deposition. The lack of clear correlations between complement activation and clinical characteristics does, at this stage, not support the use of complement inhibitors in the treatment of IgM-associated polyneuropathy.

Comparison of ELISA Versus FAMA Titers in Children After Chemotherapy and Hematopoietic Stem Cell Transplantation Who Received the Live Attenuated MAV/06 Strain Varicella Vaccine.

Varicella can lead to severe complications in immunocompromised children, including those undergoing hematopoietic stem cell transplantation (HSCT) or chemotherapy. Preventing primary varicella zoster virus (VZV) infection is crucial in these populations to mitigate morbidity and mortality. This study aimed to evaluate the immunogenicity and safety of the live attenuated MAV/06 varicella vaccine in pediatric patients post-HSCT and post-chemotherapy. Additionally, it sought to compare fluorescent-antibody-to-membrane-antigen (FAMA) and enzyme-linked immunosorbent assay (ELISA) titers to establish effective cut-off levels for protection against varicella. The FAMA assay was conducted at the Vaccine Bio Research Institute, and a VARICELLA-ZOSTER ELISA (Vircell, Granada, Spain) kit, which relies on lysate from whole cells infected with VZV, was used to determine VZV IgG. A prospective cohort study was conducted with 76 pediatric patients under 18 years old who tested negative for VZV IgG via ELISA. Patients post-HSCT and post-chemotherapy were included. Participants received the MAV/06 varicella vaccine, and serologic responses were evaluated using ELISA and FAMA. The median age of participants was 9.8 years, with acute lymphoid leukemia and acute myeloid leukemia being the most common underlying disease. Post-dose 1, the seropositive rate was 56.1% by ELISA and 97.2% by FAMA. Based on the FAMA seropositive cut-off ≥1:4, post-dose 1 geometric mean titers (GMTs) of seropositive patients in the post-HSCT group were 14.7 (95% CI, 11.3-19.1) versus 20.2 (95% CI, 13.0-31.3) in the post-chemotherapy group (p = 0.690). Based on a FAMA seropositive cut-off ≥1:16, the post-dose 1 GMT of patients considered seropositive in the post-HSCT group was 19.3 (95% CI, 15.6-24.0) versus 34.1 (95% CI, 21.0-55.4) in the post-chemotherapy group (p = 0.116), and post-dose 2 FAMA titers of 76.1 (95% CI, 14.6-398.1) in the post-HSCT group and 64.0 (95% CI, 11.4-358.1) in the post-HSCT group (p = 0.853) were observed. In patients with lower baseline FAMA titers (1:4 to 1:8), 66.7% in the post-HSCT group and 71.5% in the post-chemotherapy group achieved a greater than four-fold increase in FAMA titers post-dose 1, while those with higher baseline titers (≥1:16) did not. There were no serious adverse events or vaccine-related rashes occurring in any of the patients. The MAV/06 varicella vaccine is immunogenic in pediatric patients post-HSCT and post-chemotherapy, particularly when administered in a two-dose schedule using a cut-off FAMA titer of <1:16.

SARS-CoV-2 Antibody Seroprevalence in Gabon: Findings from a Nationwide Household Serosurvey in a Sub-Saharan Africa Country.

Seroconversion surveys of anti-SARS-CoV-2 antibodies provide accurate estimates of the prevalence of SARS-CoV-2 infections. This nationwide population-based cross-sectional serosurvey aimed to evaluate the prevalence of SARS-CoV-2 antibodies among residents in Gabon and compare the estimated cumulative number of COVID-19 cases with the officially registered number of laboratory-confirmed cases up to December 2021. Households in each province were randomly selected. Twenty-eight localities, including sixteen urban and twelve rural, were randomly selected for the study. Whole blood samples were collected in dry tubes from all study participants nationwide within 15 days. Serum samples were used to measure SARS-CoV-2-specific ELISA titers. Overall, data from 1672 households were analyzed. Out of the 3659 participants, 3175 were found to be positive for SARS-CoV-2 antibodies, resulting in a crude seroprevalence of 86.77%. Stratification of study participants by age group showed the highest seroprevalences in the 20-29 and 40-49 age groups with 91.78% (95% CI: 89.5-93.6) and 91.42% (95% CI: 88.7-93.5), respectively. Nyanga province had the lowest prevalence (72.8%), and Estuaire and Ogooué-Lolo provinces had the highest prevalence (90 and 92%). Our results suggest a high transmission rate in the Gabonese population 21 months after the first SARS-CoV-2 case in the country. This high seroprevalence estimate could indicate that the population may not have adequately implemented or appropriately adhered to the applied infection control measures.

High glutamic acid decarboxylase antibody titers may be associated with a decline in β-cell function over time and future insulin deficiency in latent autoimmune diabetes in adults

AimsLatent autoimmune diabetes in adults (LADA) is characterized by positive islet-associated autoantibodies including glutamic acid decarboxylase antibody (GADA), and gradual decline in insulin secretion, progressing to insulin dependency. This cross-sectional study aimed to determine whether GADA by enzyme-linked immunosorbent assay (GADA-ELISA) titer of ≥180 U/mL could be associated with decline in β-cell function in participants with LADA. MethodsSixty-three participants with LADA were recruited and an association between insulin secretion capacity and disease duration was investigated. Insulin peptide-specific inflammatory immunoreactivity was investigated to determine the disease’s activity. ResultsThere was a significant inverse correlation between disease duration and C-peptide index in participants with GADA-ELISA titer of ≥180 U/mL (Spearman’s r (rs) = –0.516, p < 0.01). The positivity rate of insulin peptide-specific inflammatory immunoreactivity was significantly higher in those with ≥180 U/mL than in those with <180 U/mL (p < 0.05). In participants with human leukocyte antigen (HLA)-DRB1*04:05, a significant inverse correlation was observed between disease duration and C-peptide index in those with ≥180 U/mL (rs = −0.751, p < 0.01). ConclusionsGADA-ELISA titer of ≥180 U/mL, especially with HLA-DRB1*04:05, might reflect higher disease activity and may be associated with decline in β-cell function over time and future insulin dependency in LADA.

Prokaryotic expression of N protein of akabane disease virus and preparation of monoclonal antibody

In order to generate monoclonal antibodies against the akabane virus (AKAV) N protein, this study employed a prokaryotic expression system to express the AKAV N protein. Following purification, BALB/c mice were immunized, and their splenocytes were fused with mouse myeloma cells (SP2/0) to produce hybridoma cells. The indirect ELISA method was used to screen for positive hybridoma cells. Two specific hybridoma cell lines targeting AKAV N protein, designated as 2C9 and 5E9, were isolated after three rounds of subcloning. Further characterization was conducted through ELISA, Western blotting, and indirect immunofluorescence assay (IFA). The results confirmed that the monoclonal antibodies specifically target AKAV N protein, exhibiting strong reactivity in IFA. Subtype analysis identified the heavy chain of the 2C9 mAb's as IgG2b and its light chain as κ-type; the 5E9 mAb's heavy chain was determined to be IgG1, with a κ-type light chain. Their ELISA titers reached 1:4 096 000. This study successfully developed two monoclonal antibodies targeting AKAV N protein, which lays a crucial foundation for advancing diagnostic methods for akabane disease prevention and control, as well as for studying the function of the AKAV N protein.

Evaluation Of Infectious Bursal Disease Virus Maternally Derived Antibody Decay Rate In Day Old Broilers From Different Hatcheries In Nigeria

Infectious Bursal disease (IBD) is an immunosuppressive disease of young chicks, which is responsible for major economic losses in the poultry industry worldwide, particularly for the last decades. This study utilized a cross-sectional approach to evaluate Infectious bursal disease virus (IBDV) maternally derived antibody decay rate in day old chicks (D.O.C) obtained from different hatcheries in Nigeria. Of the 450 serum samples collected through random selection with replacement method, (i.e 90 samples from the 5 hatcheries designated as farms A – E), it was observed that farm A at day 1 had mean Elisa titre of (4213 ± 366.66), farm B (2178.225 ± 292.477), farm C (1629.699 ± 229.2197), farm D (3452.609 ± 403.64.6469) and farm E (1651.789 ± 201.6811), these values were far above the protective (positive) level of 875, recommended by IDvet manual with (S/P ratios of 0.350). This level was maintained, although, with minimal decays, up to day 7 for farms A, D and E but farm B and C did not exceed day 6 and day 5 respectively. Only farm D presented protective (positive) value that lasted to day 13. The best fit for this decay rate was calculated to be Y = -2263In(x) + 3714 (week) with R2 = (0.903). From these studies we could deduce that these farms were truly vaccinated or immunized as expected and they presented different antibody titres with variable decay rates. It would have been a good idea to recommend that same vaccination schedule should not be applied indiscriminately to broilers from these farms like other researchers have, but this would rather be to the detriment of many farmers who may not be privileged to access the detailed information on which schedule is meant for which farm .We therefor recommend that broilers from these hatcheries be vaccinated between day 7 to day 10 post hatch and a booster dose (if necessary) should be administered a week after, with an intermediate plus strain vaccine type that has the capacity to penetrate through the MDA and induce humoral-immunity-in- thechicks.

IgM anti-GM2 antibodies in patients with multifocal motor neuropathy target Schwann cells and are associated with early onset.

Multifocal motor neuropathy (MMN) is a rare, chronic immune-mediated polyneuropathy characterized by asymmetric distal limb weakness. An important feature of MMN is the presence of IgM antibodies against gangliosides, in particular GM1 and less often GM2. Antibodies against GM1 bind to motor neurons (MNs) and cause damage through complement activation. The involvement of Schwann cells (SCs), expressing GM1 and GM2, in the pathogenesis of MMN is unknown. Combining the data of our 2007 and 2015 combined cross-sectional and follow-up studies in Dutch patients with MMN, we evaluated the presence of IgM antibodies against GM1 and GM2 in serum from 124 patients with MMN and investigated their binding to SCs and complement-activating properties. We also assessed the relation of IgM binding and complement deposition with clinical characteristics. Thirteen out of 124 patients (10%) had a positive ELISA titer for IgM anti-GM2. Age at onset of symptoms was significantly lower in MMN patients with anti-GM2 IgM. IgM binding to SCs correlated with IgM anti-GM2 titers. We found no correlation between IgM anti-GM2 titers and MN binding or with IgM anti-GM1 titers. IgM binding to SCs decreased upon pre-incubation of serum with soluble GM2, but not with soluble GM1. IgM anti-GM2 binding to SCs correlated with complement activation, as reflected by increased C3 fixation on SCs and C5a formation in the supernatant. Circulating IgM anti-GM2 antibodies define a subgroup of patients with MMN that has an earlier onset of disease. These antibodies probably target SCs specifically and activate complement, similarly as IgM anti-GM1 on MNs. Our data indicate that complement activation by IgM antibodies bound to SCs and MNs underlies MMN pathology.

Lymph Node Targeting Mediated by Albumin Hitchhiking of Synthetic Tn Glycolipid Leads to Robust In Vivo Antibody Production.

Tn antigen is a tumor-associated carbohydrate antigen, which is present prominently on the tumor cell surfaces and attracts an interest in vaccine development. This work demonstrates that a synthetic Tn antigen carrying glycoconjugate forms a complex with circulating albumin, delivers the antigen to lymph nodes (LNs), and leads to the efficient production of antibodies against the antigen. Synthetic Tn antigen glycoconjugate, possessing DSPE-PEG2000 linker and lipophilic moieties, undergoes micellization in PBS buffer. In the presence of bovine serum albumin (BSA), demicellization of the glycolipid occurs, with a rate constant of 0.18 min-1. In vitro studies show that the glycoconjugate binds preferentially to BSA in the presence of cells. Immunological assessments in mice models reveal the albumin-enabled delivery of the Tn glycoconjugate to antigen-presenting cells in the LNs, specifically leading to a robust humoral immune response. ELISA titers show superior binding, with a saturation dilution of 1:51 200 for Tn glycoconjugate, in comparison to that mediated by the Tn-BSA covalent conjugate with a saturation dilution of 1:6400. Immunohistochemical staining shows delivery of Tn glycoconjugate at the LNs, specifically at the subcapsular sinus and interfollicular areas. The work highlights the potential of albumin-mediated target delivery strategy for cancer immunotherapies.

Development and characterization of a novel nanobody with SRMV neutralizing activity

Peste des petits ruminants (PPR) is an acute, contact infectious disease caused by the small ruminant morbillivirus (SRMV), and its morbidity in goats and sheep can be up to 100% with significant mortality. Nanobody generated from camelid animals such as alpaca has attracted wide attention because of its unique advantages compared with conventional antibodies. The main objective of this study was to produce specific nanobodies against SRMV and identify its characteristics. To obtain the coding gene of SRMV-specific nanobodies, we first constructed an immune phage-displayed library from the VHH repertoire of alpaca that was immunized with SRMV-F and -H proteins. By using phage display technology, the target antigen-specific VHHs can be obtained after four consecutive rounds of biopanning. Results showed that the size of this VHH library was 2.26 × 1010 CFU/mL and the SRMV-F and -H specific phage particles were greatly enriched after four rounds of biopanning. The positive phage clones were selected and sequenced, and total of five independent different sequences of SRMV-specific nanobodies were identified. Subsequently, the DNA fragments of the five nanobodies were cloned into E. coli BL21(DE3), respectively, and three of them were successfully expressed and purified. Specificity and affinity towards inactivated SRMV of these purified nanobodies were then evaluated using the ELISA method. Results demonstrated that NbSRMV-1-1, NbSRMV-2-10, and NbSRMV-1-21 showed no cross-reactivity with other antigens, such as inactivated BTV, inactivated FMDV, His-tag labeled protein, and BSA. The ELISA titer of these three nanobodies against inactivated SRMV was up to 1:1000. However, only NbSRMV-1-21 displayed SRMV neutralizing activity at a maximum dilution of 1:4. The results indicate that the nanobodies against SRMV generated in this study could be useful in future applications. This study provided a novel antibody tool and laid a foundation for the treatment and detection of SRMV.

Investigation of brucellosis cases in and around Yozgat Province

Aims: Brucellosis causes a zoonotic infectious disease that is very common in our country as well as around the whole world. Infected individuals most frequently present with nonspecific symptoms such as fever, chills, malaise, and generalized body pain. Therefore, it is very difficult to diagnose. When diagnosis and treatment are delayed, it causes serious complications. It can cause pictures with high mortality, such as epididymoorchitis, endocarditis, hepatitis, neurobrucellosis, and pancytopenia. The first approach to treatment is rest. Antibiotics with intracellular bactericidal action should be used in combination due to resistance development. Methods: 136 individuals with serum titers who were diagnosed with brucellosis by serological tests and who applied to our hospital between 2017-2022 were included in the study. The patient files were analyzed to evaluate the complaints of these individuals, their Brucella ELISA titers, serum aspartate aminotransferase (AST), alanine aminotranspherase (ALT), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), lactate dehydrogenase (LDH) values, if any, at the time of diagnosis, the treatments they received, and the response to the treatment. Results: The first complaints of 136 patients who were diagnosed with brucellosis by serological tests were the most common; Joint pain was present in 48 (35.29%), widespread body pain in 26 (19.11%), and fever in 18 (13.23%). Sacroiliitis in 5 (3.67%), gonarthrosis in 4 (2.94%), bursitis in 4 (2.94%), discitis in 3 (2.2), and psoriasis in 1 of the patients’ abscess (0.73%) was detected. R=0.28 in the same direction between the first titers recognized by the patients and ALT; the same direction between titer and LDH R=0.30; the same ratio between titer and CRP is R=0.45; and there is a correlation coefficient of R=0.26 between titer and sediment in the same direction. It is important to take these correlation coefficients statistically (p&lt;0.05). Accordingly, these values also change when it titrates. But it is weakened weights as a measure of relationship. Conclusion: Although fever comes to mind first in Brucellosis, it was observed that the first complaint of individuals in our study was complicated surgery, and fever ranked third. For this reason, Brucellosis should be approached with suspicion, since it is endemic in individuals presenting with non-specific complaints. It is widely present between serum titer and ESR, and C-reactive protein as markers of inflammation. In individuals with high vibration and inflammation markers, controlled caution should be exercised. Even treatment times can be changed.

Monoglycosylated SARS-CoV-2 receptor binding domain fused with HAstem-scaffolded protein vaccine confers broad protective immunity against SARS-CoV-2 and influenza viruses

The SARS-CoV-2 and influenza pandemics have posed a devastating threat to global public health. The best strategy for preventing the further spread of these respiratory viruses worldwide is to administer a vaccine capable of targeting both viruses. Here, we show that a novel monoglycosylated vaccine designed based on the influenza virus HAstem conserved domain fused with the SARS-CoV-2 spike-RBD domain (HSSRmg) can present proper antigenicity that elicits sufficient neutralization efficacy against various SARS-CoV-2 variants while simultaneously providing broad protection against H1N1 viruses in mice. Compared with the fully glycosylated HSSR (HSSRfg), HSSRmg induced higher ELISA titers targeting HAstem and spike-RBD and exhibited significantly enhanced neutralization activity against the Wuhan pseudovirus. The enhanced immune responses raised by JR300-adjuvanted HSSRmg compared to HSSRmg alone include more anti-HAstem and anti-spike-RBD antibodies that provide cross-protection against H1N1 challenges and cross-neutralization of SARS-CoV-2 pseudoviruses. Furthermore, the enhanced immune response raised by JR300-adjuvanted-HSSRmg skews toward a more balanced Th1/Th2 response than that raised by HSSRmg alone. Notably, HSSRmg elicited more plasma B cells and memory B cells, and higher IL-4 and IFN-γ cytokine immune responses than spike (S–2P) in mice with preexisting influenza-specific immunity, suggesting that B-cell activation most likely occurs through CD4+ T-cell stimulation. This study demonstrated that HSSRmg produced using a monoglycosylation process and combined with the JR300 adjuvant elicits superior cross-strain immune responses against SARS-CoV-2 and influenza viruses in mice compared with S–2P. JR300-adjuvanted HSSRmg has great potential as a coronavirus-influenza vaccine that provides dual protection against SARS-CoV-2 and influenza infections.

Evaluation of the effectiveness of foot-and-mouth disease vaccination of animals in the buffer zone of the Republic of Armenia in 2016–2020

BackgroundFoot-and-mouth disease (FMD) is a high impact viral disease of livestock for which vaccines are extensively used for limiting the spread of infection. Armenia shares a border with both Turkey and Iran where FMD is endemic, making vaccination an important component of Armenia’s control strategy. Additionally, Armenian veterinary services utilize both passive and active monitoring for prevention control.MethodsWe sought to determine the immune status of animals vaccinated against FMD and to evaluate the effectiveness of our vaccination policy in Armenia. This was conducted in three regions including Shirak, Armavir, and Ararat Region which are located in the buffer zones that border Turkey and Iran. Through active monitoring in 2020, we studied blood serum samples from cattle and sheep using an enzyme immunoassay to determine the level of immune animals in these regions following the use of a polyvalent inactivated vaccine containing FMDV serotypes A, O, and Asia-1 that are relevant for this region. ELISA titers were assessed at 28, 90, and 180 days after vaccination in cattle of three age groups at the time of initial vaccination: 4–6 months, 6–18 months and ≥ 24 months of age with sheep of all ages.ResultsThe 3 age groups of cattle had similarly high levels of immunity with over 90% of the cattle showing a ≥ 50% protective titer 28 days after the first vaccination. By day 90, titers in cattle from the initial 4–18-month age groups dropped below 58% across the 3 serotypes and at or below 80% for the oldest cattle ≥ 24 months. Re-vaccination of cattle at 120 days did improve protective titers but never reached the level of immunity of the first vaccination. Sheep showed a similar rapid drop to less than 50% having a ≥ 50% protective titer at 90 days emphasizing the need for continual revaccination.ConclusionsThe results of this study have important implications for the current FMD vaccine policy in Armenia and improves our understanding of the rapid loss of protective titers over short periods. Since small ruminants are only vaccinated once per year and vaccination titers drop rapidly by 90 days suggests that they are vulnerable to FMD and that vaccination protocols need to be updated. Cattle should continue to be vaccinated every 3–6 months depending on their age to maintain a protective level of antibodies to protect them from FMD. More studies are needed to understand the possible role of small ruminants in the epidemiology of FMD and to evaluate revaccination at shorter intervals. These results show the concerns of rapid loss of protection to both cattle and small ruminants following 1 or more doses of commercial vaccines and that additional vaccines need to be evaluated in both groups to know how often they must be vaccinated to provide full protection. The addition of challenge studies should also be considered to better understand the level of protection as measured by serology and how it relates to protection from challenge. These results should be considered by anyone using these vaccines in cattle and sheep at longer than 3 month intervals.

Protective immunization against Enterohemorrhagic Escherichia coli and Shigella dysenteriae Type 1 by chitosan nanoparticle loaded with recombinant chimeric antigens comprising EIT and STX1B-IpaD

Increasing evidence demonstrated that Enterohemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae type 1 (S. dysenteriae1) are considered pathogens, that are connected with diarrhea and are still the greatest cause of death in children under the age of five years, worldwide. EHEC and S. dysenteriae 1 infections can be prevented and managed using a vaccination strategy against pathogen attachment stages. In this study, the chitosan nanostructures were loaded with recombinant EIT and STX1B-IpaD polypeptides. The immunogenic properties of this nano-vaccine candidate were investigated. The EIT and STX1B-IpaD recombinant proteins were heterologous expressed, purified, and confirmed by western blotting. The chitosan nanoparticles, were used to encapsulate the purified proteins. The immunogenicity of recombinant nano vaccine candidate, was examined in three groups of BalB/c mice by injection, oral delivery, and combination of oral-injection. ELISA and antibody titer, evaluated the humoral immune response. Finally, all three mice groups were challenged by two pathogens to test the ability of the nano-vaccine candidate to protect against bacterial infection. The Sereny test in guinea pigs was used to confirm the neutralizing effect of immune sera in controlling S. dysenteriae 1, infections. SDS-PAGE and western blotting, confirmed the presence and specificity of 63 and 27 kDa recombinant EIT and STX1B-IpaD, respectively. The results show that the nanoparticles containing recombinant proteins could stimulate the systemic and mucosal immune systems by producing IgG and IgA, respectively. The challenge test showed that, the candidate nano-vaccine could protect the animal model from bacterial infection. The combination of multiple recombinant proteins, carrying several epitopes and natural nanoparticles could evocate remarkable humoral and mucosal responses and improve the protection properties of synthetic antigens. Furthermore, compared with other available antigen delivery methods, using oral delivery as immune priming and injection as a booster method, could act as combinatorial methods to achieve a higher level of immunity. This approach could present an appropriate vaccine candidate against both EHEC and S. dysenteriae 1.

Correlation of desmoglein 1 and 3 immunohistochemistry with autoantibody levels and clinical severity in pemphigus.

Pemphigus is a chronic potentially fatal autoimmune bullous disorder. Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are the two common subtypes. PV is the most common and aggressive type characterized by oral mucosal erosions and cutaneous lesions. PF presents with blisters on the scalp, face, and upper trunk, and spares the mucosae. Direct immunofluorescence (DIF) is the gold standard for diagnosis. Immunohistochemistry (IHC) is an emerging alternate diagnostic tool. In this study, our objectives were to identify the staining patterns of desmoglein 1 (dsg 1) and desmoglein 3 (dsg 3) IHC and to correlate the same with autoantibody levels and clinical severity in patients with PV and PF. Forty-nine clinically, histologically, and DIF-confirmed cases of pemphigus were included in the study. The IHC patterns were scored from 0 to 3+ with 3+ dsg 1 IHC exhibiting intense membranous staining in the upper layers of the epidermis and 3+ dsg 3 IHC showing intense basal layer staining. Enzyme-linked immunosorbent assay (ELISA) for anti-dsg 1 and 3 antibodies was performed in 38 cases where serum samples were available. The pemphigus disease activity index system was utilized for clinical scoring. A 0 to 1+ score was observed for dsg 1 IHC in 100% of PF cases. A score of 0 to 1+ was observed for dsg 3 IHC in 97.3% of PV cases. One hundred percent of cases with PF and 83.9% of patients with PV tested positive for ELISA anti-dsg 1 and 3 antibody titers, respectively. Anti-dsg 1 and 3 ELISA titers significantly correlated with the dsg 1 and dsg 3 IHC scores. The mucosal scores showed a significant association with both dsg 1 and 3 IHC (p < 0.001). The cutaneous scores showed a significant association with the dsg 3 IHC (p < 0.001). The IHC patterns for dsg 1 and 3 proved reliable in giving concordant results with the ELISA antibody titers and clinical severity.

Establishment of a Mycoplasma hyorhinis challenge model in 5-week-old piglets.

Mycoplasma hyorhinis is an emerging swine pathogen with high prevalence worldwide. The main lesions caused are arthritis and polyserositis, and the clinical manifestation of the disease may result in significant economic losses due to decreased weight gain and enhanced medical costs. We aimed to compare two challenge routes to induce M. hyorhinis infection using the same clinical isolate. Five-week-old, Choice hybrid pigs were inoculated on 2 consecutive days by intravenous route (Group IV-IV) or by intravenous and intraperitoneal routes (Group IV-IP). Mock-infected animals were used as control (control group). After the challenge, the clinical signs were recorded for 28 days, after which the animals were euthanized. Gross pathological and histopathological examinations, PCR detection, isolation, and genotyping of the re-isolated Mycoplasma sp. and culture of bacteria other than Mycoplasma sp. were carried out. The ELISA test was used to detect anti-M. hyorhinis immunoglobulins in the sera of all animals. Pericarditis and polyarthritis were observed in both challenge groups; however, the serositis was more severe in Group IV-IV. Statistically significant differences were detected between the challenged groups and the control group regarding the average daily weight gain, pathological scores, and ELISA titers. Additionally, histopathological scores in Group IV-IV differed significantly from the scores in the control group. All re-isolated strains were the same or a close genetic variant of the original challenge strain. Our results indicate that both challenge routes are suitable for modeling the disease. However, due to the evoked more severe pathological lesions and the application being similar to the hypothesized natural route of infection in Group IV-IV, the two-dose intravenous challenge is recommended by the authors to induce serositis and arthritis associated with M. hyorhinis infection.

Differences between DNA vaccine and single-cycle viral vaccine in the ability of cross-protection against viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV)

Vaccination procedures can be stressful for fish and can bring severe side effects. Therefore, vaccines that can minimize the number of administrations and maximize cross-protection against multiple serotypes, genotypes, or even different species would be highly advantageous. In the present study, we investigated the cross-protective ability of two types of vaccines – viral hemorrhagic septicemia virus (VHSV) G protein-expressing DNA vaccine and G gene-deleted single-cycle VHSV genotype IVa (rVHSV-ΔG) vaccine - against both VHSV genotype Ia and infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). The results showed that rainbow trout immunized with VHSV genotype Ia G gene- or IVa G gene-expressing DNA vaccine were significantly protected against VHSV genotype Ia, but were not protected against IHNV. In contrast to the DNA vaccine, the single-cycle VHSV IVa vaccine induced significant protection against not only VHSV Ia but also IHNV. Considering no significant increase in ELISA titer and serum neutralization activity against IHNV in fish immunized with single-cycle VHSV IVa, the protection might be independent of humoral adaptive immunity. The scarcity of cytotoxic T cell epitopes between VHSV and IHNV suggested that the possibility of involvement of cytotoxic T cell-mediated cellular adaptive immunity would be low. The role of trained immunity (innate immune memory) in cross-protection should be further investigated.

Effect of Nutrients and Herbal Products on Immune Status of Broilers, Experimentally Infected with Mixed Eimeria Species

The present study was conducted to seek herbal alternatives and nutritional modulation to solve the problem of coccidiosis, a deadly protozoan disease of broilers. For the purpose, an experiment of 35 days’ duration (days 0-21 pre-infection period and days 22-35 post-infection period) was conducted to test the efficacy of herbal/natural products/dietary nutrients for the management of mixed coccidial infection prevalent under field conditions. For this purpose wheat grass juice, chelated mineral supplement, herbal anticoccidial and seeds of Embelia ribes without and with sodium bicarbonate, organic chromium, herbal vitamin E-Selenium without and with organic chromium were used to study the immunological parameters of un-infected and infected broilers with 60,000 sporulated oocysts of mixed Eimeria species. Results of the experiment revealed that LST in terms of delta optical density of T- and B-cells was maximum in wheat grass juice while ELISA titre against Newcastle Disease was maximum in herbal vitamin E-Selenium group. All the types of dietary modulation were found to be effective up to different extent in the management of poultry coccidiosis. Further research should focus on micro-array and proteomic technologies to solve the problem.

Monitoring reveals a high prevalence of Mycoplasma synoviae in layer flocks in Croatia

Mycoplasma synoviae (MS), despite its lower pathogenicity, has the ability to cause significant losses in poultry production but is usually underdiagnosed. In egg layer production, losses could be significant because of the drop in egg production and poor eggshell quality. Problems with colibacillosis and other infections secondary to MS have been continuously reported on Croatian egg layer farms. As a result, regular monitoring of flocks also included screening of the seroprevalence and molecular detection of MS using ELISA and qPCR tests, respectively. During monitoring, altogether 1135 samples were tested, including 1067 serum samples and 68 tracheal swabs, in a total of 126 flocks and 83 longitudinally merged flocks on 15 farms during the period from 2017 to 2021. The results showed a high general prevalence of MS with 86.6 % positive layer farms, while grouped flock seroprevalence and prevalence were 98.6% and 85.7%, respectively. With age, seroprevalence and ELISA titers rise significantly compared to the rearing period, with a significant mutual correlation over the entire production period. Additionally, there is a significant correlation between ELISA titers and age in weeks. Several flocks covered longitudinally from the first week of age, over the rearing period to the end of production, showed low prevalence during the rearing period, with a later significant rise in titer and prevalence, which indicates the dominance of horizontal transmission during production. Overall results indicate the need for a prompt reaction regarding preventive measures, such as better flock management, biosecurity and vaccination, which would reduce the losses and improve production.

Indirect IgG ELISA and serotype-specific neutralizing antibody titers are associated with protection against dengue in children in Cebu, Philippines

Abstract Standard measures of dengue virus (DENV) antibodies have not been established as protective against dengue. We assess whether an indirect dengue IgG ELISA (PanBio; Brisbane, QLD, Australia) and serotype-specific neutralizing antibody titers measured by a two-dilution focus reduction neutralization test (FRNT) are associated with protection against dengue. Healthy children (n = 1,214) aged 9–14 years in Cebu, Philippines were enrolled in a prospective population-based cohort study in May – June 2017 and were tested for dengue when they reported a febrile illness through March 2020. Serum samples were collected at enrollment, and all were tested by ELISA. FRNTs were performed using the WHO DENV1–4 reference strains on a random subset to quantify serotype-specific neutralizing antibodies (nAbs). Inverse probability weighting was used to adjust for subset size. The odds of dengue as predicted by antibody measures were assessed by logistic regressions and adjusted for age, gender, and residential site. Dengue naïve children were used as the reference group in all models. The probability of dengue was 9.2% (95% CI: 4.5 – 17.9) for the naïve group, 9.5% (4.8 – 17.8) for those with ELISA &amp;lt; 2, but 4.5% (2.8 – 7.2) for those with ELISA &amp;gt;= 2 (p=0.04). The probability was even lower at 1.4% (0.6 – 3.1) for those with the highest DENV1–4 geometric mean titers (GMT, p = 0.0001). Pre-vaccine nAbs to DENV2 and DENV3 were protective against a homotypic dengue case (DENV2: OR 0.28, 95% CI 0.12 to 0.65; DENV3: OR 0.27, 95% CI 0.13 to 0.57), but not heterotypic dengue. We show a commercial ELISA and serotype-specific nAbs are associated with the probability of dengue, providing useful assays for use in population and vaccine trials. This work was supported by the Philippine Department of Health, the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID), and NIH Extramural Grant: P01AI106695.