Significant Increase In Titer Research Articles

Protective antigen of Bacillus anthracis in combination with TLR4 or TLR5 agonist confers superior protection against lethal challenge in mouse model

The immunogenicity and protective ability of recombinant PA (rPA) with two innate immune system modulators, i.e., monophosphoryl lipid A (MPLA), a TLR4 agonist, and recombinant flagellin C (FliC), a TLR5 agonist, were studied in the mouse model. BALB/c mice were inoculated with three doses of rPA + alum (Alum group), rPA + FliC + alum (FliC group), rPA + MPLA + alum (MPLA group), or only alum adjuvant (Alum alone group). Significant increases in anti-PA IgG titers were observed in the Alum, FliC and MPLA groups when compared to control Alum alone group. Similarly, a significant enhancement of proinflammatory (TNF-α, IL-1β), Th1 (IFN-γ, IL-12(p70), IL-2) and Th2 (IL-10, IL-4) cytokines were also noticed in Alum, FliC and MPLA groups compared to Alum alone group. The rPA-specific IgG and cytokine responses in MPLA and FliC groups were significantly higher than the Alum group, suggesting enhancement of immune response by these TLR agonists. MPLA was also found to skew the IgG1:IgG2a ratio towards IgG2a. At a challenge dose of 25 LD50, complete protection was observed in mice of MPLA group whereas lesser protection was observed in FliC (87%) and Alum (50%) groups. Therefore, we suggest the use of MPLA in further development of rPA based anthrax vaccines.

Microbiological evaluation of probiotic therapy in patients with pulmonary tuberculosis

Patients with pulmonary tuberculosis and multiple pathogen resistance (MDR) develop persistent disorders of the intestinal microbiome during prolonged multicomponent chemotherapy requiring correction. However, there is limited data on the use of bacterial drugs in patients with tuberculosis followed by assessing their effectiveness. The aim of the study was to evaluate changes in the intestinal microbiome after a course of probiotics along with anti-tuberculosis chemotherapy in patients with MDR tuberculosis.
 Materials and methods. The design a prospective small-cohort study (n = 30). Patients with pulmonary tuberculosis received anti-tuberculosis drugs according to the IV or V regimen, the median of the doses taken was 34.5 (30; 57.5); gastrointestinal syndrome was recorded in all study subjects. Probiotic therapy was applied by using a preparation containing Bifidobacterium bifidum and B. animalis and Lactobacillus casei, L. plantarum, L. delbrueckii subsp.bulgaricus, L. acidophilus. The course of therapy comprised 21 days,1 capsule twice a day. Before and 7 days after probiotics therapy, studies on composition of the intestinal microbiota were carried out, the frequency of virulence factors Enterococcus spp., Staphylococcus spp., Candida spp. was examined; fatty acid composition and activity of enterococcal organic acid production were studied.
 Results. After a course of probiotics, a significant increase in lactobacillus titers was recorded from 5.2 (4.0; 6.0) to 6.1 (6.0; 8.0) lg CFU/g (p = 0.05). The frequency of mucosal colonization by Candida fungi and lactose-negative Escherichia decreased by 2-fold (p = 0.001) and 3-fold (p = 0.05), respectively. The frequency of detected virulent strains significantly decreased: hemolysin-producing staphylococci by 9 times (p = 0.009), enterococci with gelatinase activity by 6 times. E. faecalis membrane oleic acid level significantly increased (C9-C18:1) (p = 0.03). In E. faecium, cis-7-palmitoleic acid (C7-C16:1) and oleic (C9-C18:1) fatty acid level increased by 2-fold (p = 0.05), and for linoleic acid (C18:2) by 4 time (p = 0.04) accompanied by elevated acid formation by 1.5 times.
 Conclusion. A single course of probiotic therapy in patients with pulmonary tuberculosis leads to qualitative microbiome changes, which are characterized by decreased levels of conditionally pathogenic microorganisms with virulent properties and altered composition of the enterococcal cell membrane accompanied by their increased biochemical activity.

Phase 1 trial to model primary, secondary, and tertiary dengue using a monovalent vaccine

BackgroundThe four co-circulating and immunologically interactive dengue virus serotypes (DENV1-4) pose a unique challenge to vaccine design because sub-protective immunity can increase the risk of severe dengue disease. Existing dengue vaccines have lower efficacy in DENV seronegative individuals but higher efficacy in DENV exposed individuals. There is an urgent need to identify immunological measures that are strongly associated with protection against viral replication and disease following sequential exposure to distinct serotypes.Methods/DesignThis is a phase 1 trial wherein healthy adults with neutralizing antibodies to zero (seronegative), one non-DENV3 (heterotypic), or more than one (polytypic) DENV serotype will be vaccinated with the live attenuated DENV3 monovalent vaccine rDEN3Δ30/31-7164. We will examine how pre-vaccine host immunity influences the safety and immunogenicity of DENV3 vaccination in a non-endemic population. We hypothesize that the vaccine will be safe and well tolerated, and all groups will have a significant increase in the DENV1-4 neutralizing antibody geometric mean titer between days 0 and 28. Compared to the seronegative group, the polytypic group will have lower mean peak vaccine viremia, due to protection conferred by prior DENV exposure, while the heterotypic group will have higher mean peak viremia, due to mild enhancement. Secondary and exploratory endpoints include characterizing serological, innate, and adaptive cell responses; evaluating proviral or antiviral contributions of DENV-infected cells; and immunologically profiling the transcriptome, surface proteins, and B and T cell receptor sequences and affinities of single cells in both peripheral blood and draining lymph nodes sampled via serial image-guided fine needle aspiration.DiscussionThis trial will compare the immune responses after primary, secondary, and tertiary DENV exposure in naturally infected humans living in non-endemic areas. By evaluating dengue vaccines in a new population and modeling the induction of cross-serotypic immunity, this work may inform vaccine evaluation and broaden potential target populations.Trial RegistrationNCT05691530 registered on January 20, 2023.

Effect of humic acid and probiotics on immunity of broiler chickens.

The immune system in chickens has a fundamental role in controlling many diseases based on vaccination, thus enhancement of the immune system response is a priority. The aim of this experiment was to study the effect of probiotics and humic acid on immunity of broiler chickens. Day-old 300 Ross broiler chicks were segregated into 5 groups of 60 chicks per group. Group C was considered as a control. Groups T1, T2, T3, and T4 were given probiotics, antibiotics, humic acid for the first 7 days and humic acid for 42 days, respectively. Samples were collected on days 27 and 42 to assess the humoral immunity, cellular immunity, lymphoid organs weight, and differential leucocyte count (DLC). The results showed a significant increase (p < 0.05) in antibodies titer against Newcastle disease virus in chickens given humic acid (T4) daily for 42 days as compared to the control. There was also a significant increase in antibodies titer in the T1 group given probiotic for the first week lasting up to day 27 as compared to the control. The skin thickness of T4 group showed a significant increase as compared to T1 and T2 groups after 24 hours of DNCB challenge. After 48 hours, the thickness was still significantly higher in the T4 group as compared to other groups except for the control. There were no significant differences in Bursa of Fabricius/Body weight (%) between the groups. Spleen/Body weight (%) was significantly higher in the control group and T1 than the other groups on day 42. The DLC remains normal in all groups. It is concluded that the humic acid has a stimulant and strengthening effect on the humoral and cellular immune system when given daily to broiler chickens. Moreover, the use of humic acid and probiotics with good hygiene in the first week of age may alternate the use of antibiotics which could be toxic and raise bacterial resistance.

Effect of the third dose of BNT162b2 COVID-19 mRNA vaccine on anti-SARS-CoV-2 antibody levels in healthcare workers.

Administration of three doses of Pfizer-BioNTech BNT162b2 COVID-19 mRNA vaccine was completed in Japan in the spring of 2022. This study aimed to evaluate the antibody responses, and kinetics of three doses of vaccine in healthcare workers (HCWs). We conducted a longitudinal cohort study with HCWs, who had no history of COVID-19 or serologic evidence of SARS-CoV-2 infection, from a single hospital. Immunoglobulin G (IgG) titers of anti-SARS-CoV-2 spike protein (SP) and nucleocapsid protein (NP) titers were measured using an automated chemiluminescent enzyme immunoassay system. A total of 636 HCWs participated in the study. The anti-SP IgG titers decreased slowly after the second dose of the BNT162b2 vaccine in all participants, and robust antibody response was observed after the third dose of the vaccine. The peak anti-SP IgG titer after the third dose was approximately 4.1-fold higher than that after the first and second doses, and the rate of decrease in the anti-SP IgG titer after the third dose was significantly more gradual, than that after the second dose. After the second dose of vaccine, the antibody response was weaker in older participants than in younger participants, and in males than in females respectively, whereas the response to the third dose of vaccine did not differ significantly by sex or age. Adverse events following immunization were generally mild to moderate. The third dose of the BNT162b2 vaccine induced a significant and sustained increase in anti-SP IgG titers, and was generally safe and well-tolerated.

Effects of paraprobiotic as replacements for antibiotic on performance, immunity, gut health and carcass characteristics in broiler chickens

This study sought to determine the effects of dietary paraprobiotic (PPB) on broiler chicken performance, immunity, gut health, and carcass traits. A total of 240 day-old CARIBRO Vishal commercial broiler chicks of identical body weight randomly divided into six treatment groups, each with five replicates and eight chicks in each replicate. Six dietary treatments were preapared: T1 = (control diet), T2 = T1 + 0.02% (w/v) chlortetracycline (CTC), T3 = T1 + 0.2% (w/v) PPB, T4 = T1 + 0.4% (w/v) PPB, T5 = T1 + 0.6% (w/v) PPB and T6 = T1 + 0.8% (w/v) PPB, respectively. Body weight gain (BWG) significantly (P ≤ 0.05) increased in the T5 (0.6% PPB) and T6 (0.8% PPB) group. At the same time the feed intake significantly (P ≤ 0.05) decreased and the feed conversion ratio (FCR) significantly (P ≤ 0.05) improved in T5 and T6 group. There was a significant (P ≤ 0.05) increase in cell-mediated immunity and haem-agglutination titre (HA titre) in the 0.6% and 0.8% PPB supplemented groups compare to the control group (T1). The percentage of carcass traits and organ weights did not significantly differ between the PPB-supplemented and control groups, but the percentage of live weight in cut up parts showed a significant improvement (P ≤ 0.05) in the PPB-supplemented group. At 42 days, villus height, width, and crypt depth all significantly (P ≤ 0.05) increased in the groups supplemented with 0.6 and 0.8% para-probiotics (T5 and T6). The results show that para-probiotics can be added to broiler diets at a rate of 0.6% (w/v) to enhance performance, immunity, gut health, and breast yield. The para-probiotic may therefore be a useful substitution for antibiotic growth promoters in the diet of chickens.

Pseudomonas aeruginosa PAO1 outer membrane vesicles-diphtheria toxoid conjugate as a vaccine candidate in a murine burn model

Pseudomonas aeruginosa is an opportunistic pathogen considered a common cause of nosocomial infection with high morbidity and mortality in burn patients. Immunoprophylaxis techniques may lower the mortality rate of patients with burn wounds infected by P. aeruginosa; consequently, this may be an efficient strategy to manage infections caused by this bacterium. Several pathogenic Gram-negative bacteria like P. aeruginosa release outer membrane vesicles (OMVs), and structurally OMV consists of several antigenic components capable of generating a wide range of immune responses. Here, we evaluated the immunogenicity and efficacy of P. aeruginosa PA-OMVs (PA-OMVs) conjugated with the diphtheria toxoid (DT) formulated with alum adjuvant (PA-OMVs-DT + adj) in a mice model of burn wound infection. ELISA results showed that in the group of mice immunized with PA-OMVs-DT + adj conjugated, there was a significant increase in specific antibodies titer compared to non-conjugated PA-OMVs or control groups. In addition, the vaccination of mice with PA-OMVs-DT + adj conjugated generated greater protective effectiveness, as seen by lower bacterial loads, and eightfold decreased inflammatory cell infiltration with less tissue damage in the mice burn model compared to the control group. The opsonophagocytic killing results confirmed that humoral immune response might be critical for PA-OMVs mediated protection. These findings suggest that PA-OMV-DT conjugated might be used as a new vaccine against P. aeruginosa in burn wound infection.

Humoral Response to the Fourth BNT162b2 Vaccination and Link Between the Fourth Dose, Omicron Infection, and Disease Severity in Renal Transplant Recipients.

The effectiveness of the fourth BNT162b2 vaccination in reducing the rate and severity of coronavirus disease 2019 (COVID-19) caused by the Omicron variant in renal transplant recipients (RTRs) is unknown. Interviews were conducted with 447 RTRs regarding the status and timing of the fourth vaccination, prior vaccinations, and preceding COVID-19 infection. RTRs with polymerase chain reaction-confirmed COVID-19 infection from December 1, 2021, to the end of March 2022 were considered to have been infected with the Omicron variant and were interviewed to determine their disease severity. In a subgroup of 74 RTRs, the humoral response to the fourth dose was analyzed. In 30 RTRs, microneutralization assays were performed to reveal the humoral response to wild-type, Delta, and Omicron variant isolates before and after the fourth dose. Of 447 RTRs, 144 (32.2%) were infected with the Omicron variant, with 71 (49.3%) of the infected RTRs having received the fourth vaccine dose. RTRs who did not receive the fourth dose before the infection had more serious illness. In a subgroup of 74 RTRs, the fourth dose elicited a positive humoral response in 94.6% (70/74), with a significant increase in geometric mean titer for receptor-binding domain immunoglobulin G and neutralizing antibodies ( P < 0.001). The humoral responses to the Omicron variant before and after the fourth dose were significantly lower than the responses to the wild-type and the Delta variants. Overall, the fourth BNT162b2 dose was effective in reducing the rate and severity of Omicron disease in RTRs, despite the reduced humoral response to the variant.

Preliminary Study on the Host Response to Bivalent and Monovalent Autogenous Vaccines against Mycoplasma agalactiae in Dairy Sheep.

In Italy, dairy sheep farming represents a vital agro-industry sector, but it is still challenged by contagious agalactia (CA), which is endemic there, and vaccination is the most economical and sustainable tool for control. This study aimed to evaluate the combined Mycoplasma agalactiae (Ma)-Staphylococcus aureus (Sa) vaccine (Ma-Sa) against the Ma monovalent vaccine in ewes. Twelve primiparous Ma-free ewes were randomly grouped into three equal groups: first, the control group injected with placebo, second, the group vaccinated with the Ma monovalent vaccine, and third, the group vaccinated with Ma-Sa combined vaccine, with two S/C doses at 45-day intervals. The animals were examined for serological, hematological, and somatic cell count (SCC) changes for 17 successive weeks. A significant increase in anti-Ma antibody mean titers, leukocytes, and platelets was observed in the vaccinated animals, with the highest values in those who received the combined vaccine. Neutrophils were high only in the animals who received the combined vaccine. SCC was lower in the vaccinated animals during the first six weeks. This study concludes that the combined Ma-Sa vaccines enhance immune response and potentiate its efficacy against Ma. This improvement might be attributed to the sensitization/activation effect of S. aureus on platelets, which are recoded to act as a key regulator for the coordination of all components of the innate immune system. Even though this study included a small number of animals, its findings about the potentialities of this inactivated vaccine in the control of CA are strongly encouraging. Further confirmation might be needed through additional replicates and a challenge study is needed before proceeding with widespread use.

A switchable secrete-and-capture system enables efficient selection of Pichia pastoris clones producing high yields of Fab fragments

Pichia pastoris (syn. Komagataella phaffii) represents a commonly used expression system in the biotech industry. High clonal variation of transformants, however, typically results in a broad range of specific productivities for secreted proteins. To isolate rare clones with exceedingly high product titers, an extensive number of clones need to be screened. In contrast to high-throughput screenings of P. pastoris clones in microtiter plates, secrete-and-capture methodologies have the potential to efficiently isolate high-producer clones among millions of cells through fluorescence-activated cell sorting (FACS).Here, we describe a novel approach for the non-covalent binding of fragment antigen-binding (Fab) proteins to the cell surface for the isolation of high-producing clones. Eight different single-chain variable fragment (scFv)-based capture matrices specific for the constant part of the Fabs were fused to the Saccharomyces cerevisiae alpha-agglutinin (SAG1) anchor protein for surface display in P. pastoris. By encoding the capture matrix on an episomal plasmid harboring inherently unstable autonomously replicating sequences (ARS), this secrete-and-capture system offers a switchable scFv display. Efficient plasmid clearance upon removal of selective pressure enabled the direct use of isolated clones for subsequent Fab production. Flow-sorted clones (n = 276) displaying high amounts of Fabs showed a significant increase in median Fab titers detected in the cell-free supernatant (CFS) compared to unsorted clones (n = 276) when cells were cultivated in microtiter plates (factor in the range of ∼21–49). Fab titers of clones exhibiting the highest product titer observed for each of the two approaches were increased by up to 8-fold for the sorted clone. Improved Fab yields of sorted cells vs. unsorted cells were confirmed in an upscaled shake flask cultivation of selected candidates (factor in the range of ∼2–3). Hence, the developed display-based selection method proved to be a valuable tool for efficient clone screening in the early stages of our bioprocess development.

Improved Whole Gamma Irradiated Avian Influenza Subtype H9N2 Virus Vaccine Using Trehalose and Optimization of Vaccination Regime on Broiler Chicken

Gamma (γ)-radiation can target viral genome replication and preserve viral structural proteins compared to formalin inactivation. Thus, a stronger immunity could be induced after the inoculation of the irradiated virus. In this study, γ-irradiated low-pathogenic avian influenza virus-H9N2 (LPAIV-H9N2) was used to immunize the broiler chicken in two formulations, including γ-irradiated LPAIV-H9N2 with 20% Trehalose intranasally (IVT.IN) or γ-irradiated LPAIV-H9N2 plus Montanide oil adjuvant ISA70 subcutaneously (IV+ISA.SC) in comparison with formalin-inactivated LPAIV-H9N2 vaccine intranasally (FV.IN) or formalin-inactivated LPAIV-H9N2 plus ISA70 subcutaneously (FV+ISA.SC). Two vaccination regimes were employed; the first one was primed on day 1 and boosted on day 15 (early regime), and the second one was primed on day 11 and boosted on day 25 (late regime). A challenge test was performed with a live homologous subtype virus. Virus shedding was monitored by quantifying the viral load via RT-qPCR on tracheal and cloacal swabs. Hemagglutination inhibition (HI) antibody titration and stimulation index (SI) of the splenic lymphocyte proliferation were measured, respectively, by HI test and Cell Proliferation assay. Cytokine assay was conducted by the RT-qPCR on antigen-stimulated spleen cells. The results of the HI test showed significant increases in antibody titer in all vaccinated groups, but it was more evident in the IVT late vaccination regime, reaching 5.33 log2. The proliferation of stimulated spleen lymphocytes was upregulated more in the IVT.IN vaccine compared to other vaccines. The mRNA transcription levels of T-helper type 1 cytokines such as interferon-gamma (IFN-γ) and interleukin 2 (IL-2) were upregulated in all vaccinated groups at the late regime. Moreover, IL-6, a pro-inflammatory cytokine was upregulated as well. However, upregulation was more noticeable in the early vaccination than the late vaccination (p< 0.05). After the challenge, the monitoring of virus shedding for the H9 gene represented an extremely low viral load. The body weight loss was not significant (p > 0.05) among the vaccinated groups. In addition, the viral load of <100.5 TCID50/ml in the vaccinated chicken indicated the protective response for all the vaccines. Accordingly, the IVT vaccine is a good candidate for the immunization of broiler chicken via the intranasal route at late regime.

Lung Transplant Recipients Immunogenicity after Heterologous ChAdOx1 nCoV-19-BNT162b2 mRNA Vaccination.

(1) Background: High immunosuppressive regimen in lung transplant recipients (LTRs) hampers the immune response to vaccination. We prospectively investigated the immunogenicity of heterologous ChAdOx1 nCoV-19-BNT162b2 mRNA vaccination in an LTR cohort. (2) Methods: Forty-nine COVID-19 naïve LTRs received a two-dose regimen ChAdOx1 nCoV-19 vaccine. A subset of 32 patients received a booster dose of BNT162b2 mRNA vaccine 18 weeks after the second dose. (3) Results: Two-doses of ChAdOx1 nCoV-19 induced poor immunogenicity with 7.2% seropositivity at day 180 and low neutralizing capacities. The BNT162b2 mRNA vaccine induced significant increases in IgG titers with means of 197.8 binding antibody units per milliliter (BAU/mL) (95% CI 0–491.4) and neutralizing antibodies, with means of 76.6 AU/mL (95% CI 0–159.6). At day 238, 32.2% of LTRs seroconverted after the booster dose. Seroneutralization capacities against Delta and Omicron variants were found in only 13 and 9 LTRs, respectively. Mycophenolate mofetil and high-dose corticosteroids were associated with a weak serological response. (4) Conclusions: The immunogenicity of a two-dose ChAdOx1 nCoV-19 vaccine regimen was very poor in LTRs, but was significantly enhanced after the booster dose in one-third of LTRs. In immunocompromised individuals, the administration of a fourth dose may be considered to increase the immune response against SARS-CoV-2.

Neutralizing IgG antibody seroprevalence, efficacy and safety post covishield vaccination

COVID-19, a pandemic caused by severe acute respiratory syndrome (SARS) corona virus-2 has been a worldwide challenge for the entire mankind. However, a safe and efficacious vaccine would be clinically valuable to reduce the risks of COVID-19. In this study, we aimed to assess the seroprevalence of the Neutralizing IgG Antibodies six months after completion of both the doses of Covishield Vaccination (ChAdox1 nCoV-19) and compare it with the IgG titres one month after the first dose and one month after the second dose of the vaccine respectively. The efficacy, safety and comorbidities related to the vaccine were also assessed at different time intervals. The levels of IgG antibodies were estimated in 72 subjects from the Teerthanker Mahaveer Medical College &amp; Research Centre (who turned up after six months of the complete regimen of Covishield Vaccination) using the Enzyme-Linked Immunosorbent Assay (ELISA) Technique. A highly significant increase (p= 0.00) in the neutralizing Antibodies titre was seen in fully vaccinated individuals post six months when compared to the titres of post one month of 1st &amp; 2nddoses. It can therefore be concluded that (ChAdox1 nCoV-19) Covishield Vaccination if administered in full regimen has both acceptable efficacies as well as safety profile.

#40 An RSV live-attenuated vaccine candidate lacking G protein mucin domains is highly attenuated, immunogenic, and effective in RSV prevention in BALB/c mice

Abstract Background RSV is a leading viral respiratory pathogen in infants. Live attenuated vaccines (LAV) show promise to reduce the disease burden of RSV disease. The RSV attachment protein (G protein) is heavily glycosylated with mucin domains that promote viral attachment. They may also aid in immune evasion through steric shielding of immunogenic epitopes. The objective of this study was to create and rescue RSV LAV candidates lacking the RSV G protein mucin domains with attenuated LAV replication and increased immunogenicity. Method LAV vaccine candidates “G155” and “G155-S” were generated from recombinant A2-line19F strain RSV by deletion of the G-protein mucin domains. G155-S additionally underwent deletion of the transmembrane domain, such that G155-S only expressed the secreted form of the G protein, while G155 expressed both transmembrane and secreted forms. Western blotting was used to characterize F and G protein expression in both vaccine candidates in comparison to wild-type (wt) A2-line19F. Enzyme-linked immunosorbent assays (ELISAs) were used to measure monoclonal antibody binding to LAV virus lysate. Viral growth kinetics were measured in HEp2 cells and in BALB/c mouse lungs by a fluorescent focus unit (FFU) assay. Immunogenicity was determined by measuring serum neutralizing antibody titers in BALB/c mice following a prime/boost regimen on days 0 and 28. Mice were challenged with wt RSV on day 60, and LAV efficacy was determined by measuring RSV lung viral loads by FFU assay and RT-PCR on day 4 post-RSV challenge. Results The vaccine candidates showed increased monoclonal antibody binding to surface F protein by ELISA, despite similar levels of F protein expression to wt, consistent with F epitope de-shielding. Both vaccine candidates were significantly attenuated in vitro and were undetectable in mouse lungs via FFU assay. By RT-PCR, LAV viral RNA was reduced 3-fold for G155-S, and 22-fold for G155 compared to wt RSV in mouse lungs. Both vaccine candidates elicited similar neutralizing antibody titers compared to wt A2-line19F on day 28 (G155 GMT 13, 95% CI 3, 53; G155-S GMT 10, 95% CI 2, 51; A2-line19F GMT 13, 95% CI 12, 15), and significant increases in titers were observed on day 59 (G155 GMT 440, 95% CI 5, 36057; G155-S 436, 95% CI 354, 537; A2-line19F 275, 95% CI 134, 567). Following RSV challenge, vaccinated mice showed no detectable lung virus by FFU assay and a reduction in lung viral RNA by RT-PCR of 560-fold for G155 and 604-fold for G155-S compared to RSV-challenged unvaccinated mice. Conclusion G155 and G155-S are highly attenuated RSV LAV candidates that protect against RSV challenge in mice and produce high levels of neutralizing antibodies comparable to wt RSV.

Immunomodulatory Activity of a Polyherbal Immune Blend in Wistar Rats

Abstract Objectives The immune system plays an important role in protection against foreign organisms and its modulation is implicated in infection, inflammatory and autoimmune diseases. Several plant extracts have been explored as immune modulators which work either as immunosuppressive or immune-stimulating agents. he present study explores an in vivo immunomodulatory activity of an in-house formulated combination of plant extracts, called as Immune blend consisting of extracts of Boswellia, Ashwagandha, Neem, Ultrasol Curcumin, and Star anise. Methods The study was performed using 32 male wistar rats divided into 4 groups (vehicle control, dexamethasone, immune blend, dexamethasone + immune blend). Immunosuppression was induced in rats using dexamethasone (5 mg/kg body weight) intraperitoneally. The in-vivo immunomodulatory activity of Immune blend (at a dose of 50 mg/kg body weight) was evaluated by measurement of hemagglutination titer, delayed-type hypersensitivity (DTH) response against a foreign antigen and carbon clearance test. Additionally, synergy of the combination of plant extracts was measured in lipopolysaccharide (LPS) -induced THP1 cells for their ability to inhibit inflammatory cytokines through fluorescence-activated cell-sorting (FACS). Results Immune blend demonstrated significant (P &amp;lt; 0.05) increase in hemagglutination titer against sheep red blood cell (SRBC) as compared to dexamethasone control animals. Further immune blend also led to significant increase (P &amp;lt; 0.05) in cell mediated immune response against SRBC as measured by foot paw edema as compared to dexamethasone control animals. Pretreatment with the immune blend also showed a significant rise (P &amp;lt; 0.01) in phagocytic index indicated by phagocytic activity. Immune blend significantly inhibited expression of TNF-α and IL-2 in LPS-stimulated THP1 cells with synergistic response with the blend as compared to individual ingredients. Conclusions The study revealed that the immune blend stimulates both the cellular and the humoral immune responses under normal and immunosuppressed conditions in wistar rat model suggesting its therapeutic usefulness. Funding Sources OmniActive Health Technologies Limited.

Deficiency of antibody-suppressor CXCR5+CD8+ T cells (not CD4+ Tregs) drives high alloantibody production in CCR5 KO kidney transplant recipients

Abstract Kidney transplant (KTx) into CCR5 KO mice is an excellent model to study AMR immunobiology due to a heightened humoral response posited to be secondary to impaired CD4+Treg cell trafficking to the allograft. We have observed that adoptive cell therapy (ACT) with alloprimed CXCR5+CD8+ T cells mediates significant reduction of alloAb titer in CCR5 KO KTx recipients. We hypothesized that CXCR5+CD8+ T cells are more potent inhibitors of alloAb production than CD4+Tregs. We found that CCR5 KO recipients have significantly fewer CXCR5+CD8+ T cells (782±183 vs 2058±167 cells/mm3) and graft-infiltrating CD4+Treg cells (CD25+FoxP3+; 12.9±8.8 vs 56.2±8.8 cells/mm3) compared to WT recipients, as well as significantly greater quantity of splenic germinal center (GC; GL7+Fas+B220+) B cells (5814±436 vs 671±342 cells/mm3; all p&amp;lt;0.003). CCR5 KO recipient alloAb titer is significantly reduced following CXCR5+CD8+ T cell ACT (1259±436 vs 5951±404 in untreated controls; p&amp;lt;0.0001) but not following CD4+Treg ACT (5688±436). CXCR5+CD8+ T cells reduced splenic GC B cell (5813±589 to 4087±537; p=0.002) quantity, while CD4+Treg had no impact (6916±538/mm3). Compared to untreated controls, CD8-depletion in WT recipients led to significant increase in alloAb titer (1508±338 vs 6078±436) and GC B cells (671±436 vs 7761±430; p&amp;lt;0.0001 for both). CD4+Treg depletion led to increased alloAb titer (3180±436) and GC B cell quantity (4911±429; p&amp;lt;0.004 for both), though less pronounced than in the CD8-depleted group (both p&amp;lt;0.001). This suggests that CXCR5+CD8+ T cells are more potent regulators of alloAb production than CD4+Tregs and their deficiency in CCR5 KO recipients is the major driver of heightened humoral immunity and AMR following KTx. Supported by NIH R01 AI083456 (to GLB), T32 AI106704-07 and F32 AI161844 (to JLH), CA016058, UL1TR002733, the OSU Division of Transplant Surgery, and the OSU College of Medicine.

Humoral Response to the Third Dose of Sars-Cov-2 Vaccine in Kidney Transplant Recipients

BackgroundMost solid organ transplant recipients did not develop an appreciable serologic response after 2 doses of the mRNA SARS-CoV-2 vaccine.MethodsWe analyzed the humoral response after a third dose of the BNT162b2 vaccine in 130 kidney transplant recipients, compared to 48 health care workers, and associated factors, including prevaccine cellular immune response, by evaluating intracellular cytokine production after stimulation of donor's peripheral blood mononuclear cells.ResultsAfter 2 doses, most of the controls (47 out of 48, 98%) and only 40% of kidney recipients (52 of 130) kidney recipients were seropositive (P < .001). Most seronegative recipients developed a serologic response after the booster (47 out 78, 60%), thus bringing the total number of seropositive recipients to 99 out of 130 (76%). After the third dose, there was a significant increase in antibodies titers in both groups. Decreased humoral response was significantly associated with an older age, lower lymphocyte count, and a lower level of antibodies before booster administration. CD4+TNFα+ and CD4+INFγ+ were correlated with mean increase in antibody titers.ConclusionsA third dose of the BNT162b2 mRNA vaccine in kidney recipients is safe and effectively results in increased IgG anti-S levels, including in individuals who were seronegative after 2 doses. Long-term studies of the length of the immune response and protection are required.

Scale-up issues for commercial depth filters in bioprocessing.

Significant increases in cell density and product titer have led to renewed interest in the application of depth filtration for initial clarification of cell culture fluid in antibody production. The performance of these depth filters will depend on the local pressure and velocity distribution within the filter capsule, but these are very difficult to probe experimentally, leading to challenges in both process design and scale-up. We have used a combination of carefully designed experimental studies and computational fluid dynamics (CFD) to examine these issues in both lab-scale (SupracapTM 50) and pilot-scale (StaxTM ) depth filter modules, both employing dual-layer lenticular PDH4 media containing diatomaceous earth. The SupracapTM 50 showed a more rapid increase in transmembrane pressure and a more rapid DNA breakthrough during filtration of a Chinese Hamster Ovary cell culture fluid. These results were explained using CFD calculations which showed very different flow distributions within the modules. CFD predictions were further validated using measurements of the residence time distribution and dye binding in both the lab-scale and pilot-plant modules. These results provide important insights into the factors controlling the performance and scale-up of these commercially important depth filters as well as a framework that can be broadly applied to develop more effective depth filters and depth filtration processes.

Long-Term Varicella Zoster Virus Immunity in Paediatric Liver Transplant Patients Can Be Achieved by Booster Vaccinations-A Single-Centre, Retrospective, Observational Analysis.

Varicella is one of the most common vaccine-preventable infections after paediatric solid organ transplantation; thus, vaccination offers simple and cheap protection. However, children with liver disease often progress to liver transplantation (LT) before they reach the recommended vaccination age. As a live vaccine, varicella zoster virus (VZV) vaccination after transplantation is controversial; however, many case series demonstrate that vaccination may be safe and effective in paediatric liver transplant recipients. Only limited data exists describing long-term vaccination response in such immunocompromised patients. We investigated retrospectively vaccination response in paediatric patients before and after transplantation and describe long-term immunity over ten years, including the influence of booster-vaccinations. In this retrospective, single-centre study, 458 LT recipients were analysed between September 2004 and June 2021. Of these, 53 were re-transplantations. Patients with no available vaccination records and with a history of post-transplant lymphoproliferative disease, after hematopoietic stem cell transplantation and clinical chickenpox were excluded from this analysis (n = 198). In total, data on 207 children with a median annual follow-up of 6.2 years was available: 95 patients (45.9%) were unvaccinated prior to LT. Compared to healthy children, the response to vaccination, measured by seroconversion, is weaker in children with liver disease: almost 70% after one vaccination and 93% after two vaccinations. One year after transplantation, the mean titres and the number of children with protective antibody levels (VZV IgG ≥ 50 IU/L) decreased from 77.5% to 41.3%. Neither diagnosis, gender, nor age were predictors of vaccination response. Booster-vaccination was recommended for children after seroreversion using annual titre measurements and led to a significant increase in mean titre and number of protected children. Response to vaccination shows no difference from monotherapy with a calcineurin inhibitor to intensified immunosuppression by adding prednisolone or mycophenolate mofetil. Children with liver disease show weaker seroconversion rates to VZV vaccination compared to healthy children. Therefore, VZV-naïve children should receive basic immunization with two vaccine doses as well as those vaccinated only once before transplantation. An average of 2–3 vaccine doses are required in order to achieve a long-term seroconversion and protective antibody levels in 95% of children.

1193. Mucosal Antibody Responses to Respiratory Syncytial Virus (RSV) in Infants and Young Children

Abstract Background The burden associated with RSV infection is substantial. Although RSV initially infects the upper respiratory tract, there is limited information of the mucosal concentrations of antibodies (Abs) directed to RSV specific proteins and whether patient’s age and disease severity influence production of mucosal Abs. Methods From 2017 to2019 we enrolled previously healthy children &amp;lt; 2 years of age hospitalized with RSV infection and obtained acute and convalescent (day; D30) nasopharyngeal (NP) samples to measure preF and postF specific IgG and IgA Abs by ELISA. Demographic and clinical data were collected and analyzed according to Abs responses. Results We enrolled 77 children (median [IQR] age: 2.8 [1.5-5.2] months; 49 % females) within the first 24 hours of hospitalization. Of those 25 (33%) patients required PICU care. A significant increase in convalescent IgG preF Abs titers was detected in 62 (81%) children, while IgA preF titers significantly increased in all but one child on D30. The magnitude of the increase was 56-fold higher for preF IgA versus preF IgG (p&amp;lt; 0.0001). PostF IgG and IgA titers were also increased on D30 but at significant lower levels. Infants &amp;lt; 3 months of age compared with those &amp;gt;3-24 months had significantly higher baseline preF and postF IgG Abs titers (p &amp;lt; 0.001) but not IgA antibodies. D30 preF and post F IgG titers were higher in children &amp;gt; 6 months of age (p &amp;lt; 0.0001) but only preF titers fold change significantly correlated with age (r=0.4, p&amp;lt; 0.0001). These correlations were not identified with IgA preF antibodies. There were no statistical differences in antibody titers at baseline and on D30 according to breastfeeding, and disease severity as defined by the need for PICU care. Conclusion Children hospitalized with RSV infection demonstrated significantly increased titers of mucosal preF and post F IgG and IgA specific Abs in convalescent samples. Baseline IgG Abs where higher in younger infants, which likely reflects maternally acquired antibodies. Age significantly correlated with Abs production, suggesting a more robust immune response in older children. Disclosures Mark E. Peeples, PhD, Janssen (Research Grant or Support)Pfizer (Research Grant or Support) Octavio Ramilo, MD, Adagio (Consultant)Bill &amp; Melinda Gates Foundation (Grant/Research Support)Janssen (Grant/Research Support)Lilly (Consultant)Merck (Consultant, Grant/Research Support)NIH (Grant/Research Support)Pfizer (Consultant)SANOFI (Board Member) Asuncion Mejias, MD, PhD, MsCS, Janssen (Grant/Research Support, Advisor or Review Panel member)Merck (Grant/Research Support, Advisor or Review Panel member)Roche (Advisor or Review Panel member)Sanofi (Advisor or Review Panel member)