Measurement Of Antibody Levels Research Articles

Early Antibody Temporal Responses to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Vaccinated Subjects Determined by the cobas 6000 Spike Assay.

To the Editor.—Three coronavirus disease 2019 (COVID-19) vaccines targeting the viral spike protein (S)1 have received US Food and Drug Administration (FDA) Emergency Use Authorization (EUA).2 We measured levels of anti–severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in adults after they received Moderna, Pfizer-BioNTech (Pfizer), or Janssen/Johnson & Johnson (J&J) vaccines.Between March and May 2021, we collected blood from 60 adults who had received at least 1 of 2 doses of Moderna (n = 48) or Pfizer (n = 6) vaccines or 1 dose of the J&J (n = 6) vaccine. Use of the samples was deemed exempt by the National Institutes of Health Institutional Review Board.Plasma was analyzed for antibodies using Roche anti–SARS-CoV-2-S and anti–SARS-CoV-2-nucleocapsid (N) immunoassays on a cobas-6000 analyzer. Both assays have FDA EUAs and measure immunoglobulin (Ig) G, IgA, and IgM against their target proteins. Twelve J&J specimens were additionally analyzed for anti-S using a luciferase immunoprecipitation system assay.3Participants had a median age of 46.2 years (range, 18–64 years) and 36 of the 60 participants (60%) were women. All Moderna (n = 48) vaccine recipients and all but 1 Pfizer vaccine recipients (n = 5) were positive for anti-S (Figure, A). This included 5 Moderna participants and 1 Pfizer participant who had received only 1 dose. All anti-N results were negative, except for 4 participants with history of SARS-CoV-2 infection (Figure, A).Moderna and Pfizer vaccines induced positive anti-S as early as 7 and 19 days, respectively, post–first shot. A Pfizer participant with sequential samples was negative at day 27 and positive at day 84 (Figure, B). In J&J vaccine recipients (Figure, C), 2 participants (participant [P] 1 and P2) negative at days 16 (P1) and 19 (P2) became positive by days 20 and 30, respectively. P3 and P4 were positive when first tested at days 19 and 20 postvaccine. All 4 participants showed increases in anti-S levels, reaching 250 U/mL or more and 227.6 U/mL or more in P2 and P3, respectively, on day 69. Two participants, P5 (positive) and P6 (negative), were only tested once, at day 26. The 12 J&J specimens analyzed with a different assay showed even fewer specimens positive for anti-S (n = 6) than the Roche assay (n = 9) (Figure, D).We found that nearly all vaccinated Moderna participants had anti-S levels that exceeded the upper limit of the Roche assay. By contrast, J&J vaccine recipients had anti-S levels that were negative or low 2 weeks after vaccination and gradually increased over time. Our results suggest that if J&J vaccine recipients were tested 2 weeks after vaccination, many may erroneously have been considered vaccine failures. It is possible that the commercial assay is insensitive, but there were even fewer positives with the immunoprecipitation assay. Alternatively, the J&J vaccine may induce an effective amnestic response, and/or another antibody or T-cell response may correlate with efficacy. Of note, the highly effective varicella vaccine induces antibody responses that are often not detected using commercial assays, and thus it is recommended that vaccinated patients not get tested.4 Our study is limited by the number of participants, particularly those who had received the Pfizer vaccine, which does not allow for strong conclusions regarding the similarities with or differences from Moderna or J&J in antibody temporal responses. Despite this limitation, the repeated testing of J&J participants indicates that measuring antibody levels within the first weeks of vaccination may give negative results and lead to unwarranted additional doses of vaccine.

Antibodies and protection against COVID-19

Antibodies are one of the most important components of the immune system against infections. So far, little is known about the contribution of antibodies in the protection against the SARS-CoV-2. Measuring antibody levels in the population will allow to determine who has been infected; this could also be useful to know if people with certain levels of antibodies are already protected against the disease.

Affinity of Anti-Spike Antibodies in SARS-CoV-2 Patient Plasma and Its Effect on COVID-19 Antibody Assays

Background: Measuring anti-spike protein antibodies in human plasma or serum is commonly used to determine prior exposure to SARS-CoV-2 infection and to assess the anti-viral protection capacity. According to the mass-action law, a lesser concentration of tightly binding antibody can produce the same quantity of antibody-antigen complexes as higher concentrations of lower affinity antibody. Thus, measurements of antibody levels reflect both affinity and concentration. These two fundamental parameters cannot be disentangled in clinical immunoassays, and so produce a bias which depends on the assay format. Methods: To determine the apparent affinity of anti-spike protein antibodies, a small number of antigen-coated magnetic microparticles were imaged by fluorescence microscopy after probing antigen-antibody equilibria directly in patient plasma. Direct and indirect anti-SARS-CoV-2 immunoassays were used to measure antibody levels in the blood of infected and immunized individuals. Findings: We observed affinity maturation of antibodies in convalescent and vaccinated individuals, showing that higher affinities are achieved much faster by vaccination. We demonstrate that direct and indirect immunoassays for measuring anti-spike protein antibodies depend differently on antibody affinity which, in turn, affects accurate interpretation of the results. Interpretation: Direct immunoassays show substantial antibody affinity dependence. This makes them useful for identifying past SARS-CoV-2 exposure. Indirect immunoassays provide more accurate quantifications of anti-viral antibody levels. Funding: No external funding sources were used in this study.Declaration of Interest: All authors are employed by Abbott LabsEthical Approval: Patient plasma/serum samples used for this study were purchased from New York Biologics (Southampton, NY), Access Biologicals, LLC (Vista, CA) and New York Blood Center (New York, NY). The samples purchased from Access Biologicals LLC were collected under an IRB protocol approved by Ballad Health System IRB. The samples purchased from New York Biologics were collected under an IRB approved by Ethical and Independent Review Services (E&I). The patient plasma samples purchased from New York Blood Center (New York, NY), were collected from volunteer blood donors who consented to the use of their samples for research purposes at the time of collection.

The immunoglobulin G antibody response to malaria merozoite antigens in asymptomatic children co-infected with malaria and intestinal parasites.

Co-infection with malaria and intestinal parasites is common in children in Africa and may affect their immune response to a malaria parasite infection. Prior studies suggest that co-infections may lead to increased susceptibility to malaria infection and disease severity; however, other studies have shown the reverse. Knowledge on how co-morbidities specifically affect the immune response to malaria antigens is limited. Therefore, this study sought to determine the prevalence of co-infection of malaria and intestinal parasites and its association with antibody levels to malaria merozoite antigens. A cross sectional study was carried out in two villages with high transmission of malaria in Cameroon (Ngali II and Mfou) where mass drug administration (MDA) had been administered at ~6-month intervals (generally with albendazole or mebendazole). Children aged 1-15 years were enrolled after obtaining parental consent. A malaria rapid diagnostic test was used on site. Four (4) ml of peripheral blood was collected from each participant to determine Plasmodium falciparum infections by microscopy, haemoglobin levels and serology. Fresh stool samples were collected and examined by wet mount, Kato-Katz method and modified Ritchie concentration techniques. A Multiplex Analyte Platform assay was used to measure antibody levels. A total of 320 children were enrolled. The prevalence of malaria by blood smear was 76.3% (244/320) and prevalence of malaria and intestinal parasites was 16.9% (54/320). Malaria prevalence was highest in young children; whereas, intestinal parasites (IP+) were not present until after 3 years of age. All children positive for malaria had antibodies to MSP142, MSP2, MSP3 and EBA175. No difference in antibody levels in children with malaria-co infections compared to malaria alone were found, except for antibody levels to EBA-175 were higher in children co-infected with intestinal protozoa (p = 0.018), especially those with Entamoeba histolytica infections (p = 0.0026). Antibody levels to EBA175 were significantly higher in children co-infected with malaria and E. histolytica compared to children infected with malaria alone. It is important to further investigate why and how the presence of these protozoans might modulate the immune response to malaria antigens.

Rapid response platform to manufacture human immunoglobulins

Abstract A rapidly deployable drug product for use in the early stages of a disease outbreak could prevent further spread of the pathogen. Emergent BioSolutions is developing a rapid response platform (RRP) to manufacture human immunoglobulins (HIG) for use as a passive immunotherapy during public health emergencies. Passive immunotherapies derived from either convalescent plasma or purified immunoglobulins (Ig) have a long history of success in treating a range of viral, bacterial and toxin diseases, as they provide the immediate benefit of a protective response. Emergent's RRP is being developed to address the need for front-line therapy/prophylaxis with immediate benefit with the potential to overcome the current limitations of convalescent plasma. Emergent's proposed RRP begins with identification of plasma donors with measurable levels of pathogen-specific antibodies using a field deployable real-time donor screening assay. Plasma collected from eligible donors is treated using the Mirasol® Pathogen Reduction Technology, pooled, purified and concentrated into HIG using Emergent's rapid HIG manufacturing process to produce product formulated for intramuscular administration. Our rapid HIG process is chromatography based and each 30 L run aims to purify, formulate, and fill ∼65 doses (1 g of total IgG) with testing and release onsite for immediate use. Multiple innovations to streamline operations, including single-use technologies and minimal human interfaces, allow for the manufacture of HIG product in ∼30 hours. To house the process, Emergent has designed and constructed a working modular manufacturing unit (MMU) prototype. The MMU consists of a modified 53-foot shipping container designed for multiple modes of transport, to meet ISO 8 environment requirements with self-contained utilities, including HVAC, and can run off a diesel generator or local power. Product from multiple development runs within the MMU have been characterized and validation work is ongoing. Key messages A modular manufacturing unit is being developed with the potential to provide a local capability to produce health solutions for endemic diseases. A modular manufacturing unit is being developed with the potential to rapidly manufacture human immunoglobulins during public health emergencies and pandemics.

Protective immunity induced by a DNA vaccine cocktail expressing TgSAG1, TgROP2, and the genetic adjuvant HBsAg against Toxoplasma gondii infection

Toxoplasma gondii is an intracellular obligate parasitic protozoon that can infect all warm-blooded animals, causing zoonotic toxoplasmosis. So far, there is no commercial toxoplasmosis vaccine for human use. In the present study, we constructed a DNA vaccine cocktail which includes the surface protein (SAG1) and the rhoptry protein ROP2 denoted as pEGFP-N1-SAG1-ROP2. In order to improve the efficacy, HBsAg was used as a genetic adjuvant to construct pEGFP-N1-HBsAg-SAG1-ROP2. Two eukaryotic plasmids were transiently transfected into HEK293T cells and the expression was examined using fluorescence microscopy and western blotting. We then immunized Kunming mice intramuscularly with the DNA vaccine. After three immunizations, the immune response was evaluated by measuring antibody levels, cytokine production, percentages of CD4+ and CD8+ T lymphocytes, and the survival times of the T. gondii RH strain challenged mice. The results showed that the two DNA vaccines stimulated Th1 responses, and had a higher antibody titer, IL-2, IL-12, and IFN-γ levels, and percentage of CD4+ and CD8+ T lymphocytes than the control group. In addition, mice immunized with the pEGFP-N1-HBsAg-SAG1-ROP2 vaccine showed increased survival times compared with pEGFP-N1-SAG1-ROP2.

COVID-19 convalescent plasma: phase 2.

See editorial on page 1123–1127, in this issue

Low exposure to Plasmodium falciparum and Acquisition of Antibodies to Circumsporozoite Protein Antigens in Individuals Living in Western Highlands of Kenya with Unstable Malaria Transmission

Background: Malaria continues to be a major public health concern despite the concerted efforts to eliminate it. Antibodies to Plasmodium falciparum (P. falciparum) antigens are involved in prevention of infection and disease with some of the antigens being targeted as lead candidates in vaccine development. However, most of the studies have been done in malaria endemic areas with little information available on exposure and acquisition of protective antibodies in areas of low and unstable malaria transmission. This study sought to determine whether the extent of exposure to P. falciparum affect acquisition of protective antibodies by measuring antibody levels and determine the prevalence of circumsporozoite protein (CSP) and crude schizont extract (SE) in individuals living in an area with low unstable malaria transmission in Western Highlands of Kenya.Methods: Sixty plasma samples from individuals living in an area of low unstable malaria transmission in Western Highlands of Kenya were randomly selected and categorized into three age groups: 18years (n=14). Antibodies levels in plasma were measured by Enzyme Linked Immunosorbent Assay (ELISA) and compared to known positive and negative control plasma samples. Prevalence and levels of antibodies produced against circumsporozoite protein and schizont extract antigens were compared between age groups.Results. The prevalence of antibodies ranged from 0% to 14.29% at arbitrary units (AU)>2 for the two antigens. The antibody prevalence did not significantly increase with age (P>0.05) but correlated with CSP and SE antigens (r=0.5977; P<0.05).Conclusion This study highlights antibody responses to CSP and SE antigens in individuals living in low and unstable malaria transmission. The levels of antibodies were generally low across all age groups and there were no significant differences among age groups. Longitudinal study on more antigens is needed to inform exploration of multi-antigen vaccines and also adopt several control measures including Epidemic Preparedness and Response (EPR).

Effectiveness of vaccination against influenza in HIV-infected pediatric patients in the 2016-2017 epidemic season.

Pediatric patients infected with the human immunodeficiency virus (HIV) are at risk of developing severe influenza. Vaccination against influenza in HIV (+) children in Poland is recommended and free of charge. The aim of the study was to evaluate the effectiveness of influenza vaccination in this group of patients. The study group consisted of 25 HIV-infected children, patients of the Department of Paediatric Infectious Diseases in Wrocław, vaccinated against influenza from September to December 2016. The incidence of influenza and influenza-like diseases was monitored from December 1, 2016 to April 15, 2017. IgG / IgM against influenza A and B by ELISA and against AH1N1 by HI method before vaccination and 4-6 weeks after vaccination were determined. Nasopharyngeal swabs of influenza RNA for PCR testing were collected in each patient during each hospitalization in the epidemic season. In the 2016/2017 epidemic season, none of the patients developed symptomatic influenza or any other flu-like infection with fever. Influenza A virus RNA was detected in nasopharyngeal swabs in 8 patients, none of them presented any clinical symptoms of infection. Positive pre-vaccination IgG levels against influenza A and B were detected in 36% (9/25) and 68% (17/25) of patients, respectively. After vaccination, positive concentrations were found in 56% (14/25) and 80% (20/25) of patients, respectively. Positive concentration of IgM antibodies was achieved by 60% (15/25) of the respondents - for influenza A and 52% (13/25) - for influenza B. In 11 patients (45.8%) before vaccination, protective titres of antibodies against H1 haemagglutinin were obtained, after the vaccination the proportion of patients with a protective titer was 52.4%. Influenza vaccination in HIV-infected children is effective in preventing symptomatic influenza virus infection but does not prevent transmission of infection in the population. Annual influenza vaccination and seasonal natural infections with influenza viruses result in the persistence of measurable antibody levels until the next epidemic season.

A Strategy to Battle Coronavirus Disease 2019 in the Hospital: Identify and Use the Power of "Immune Survivors".

The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses serious health and economic problems worldwide. One of the worst scenarios is the collapse of the medical care system due to nosocomial infections. SARS-CoV-2 quickly spreads in closed spaces, crowded areas, and close physical distances, which frequently occur in Japanese medical facilities. Although we are making efforts to avoid such situations, healthcare workers always face the risk of developing a SARS-CoV-2 infection in the workplace because of proximity. Thus, we need to battle SARS-CoV-2 using a unique strategy.We propose a novel strategy to eliminate SARS-CoV-2 infections: measurement of antibodies against SARS-CoV-2 and using the power of “immune survivors.” We agree with using standard precautions and early isolation of patients with coronavirus disease 2019 (COVID-19) to block the spread of SARS-CoV-2 infection. However, we face difficulties carrying out these fundamental missions. Now, we focus on “immune survivors.” If healthcare workers acquired the neutralizing antibody against SARS-CoV-2, they are considered “immune survivors” with a low risk of reinfection with SARS-CoV-2. These “immune survivors” can contribute to the care of patients with COVID-19 on the front line. Also, these “immune survivors” can function as an envelope by surrounding COVID-19 patients. As a result, “immune survivors” can eliminate the spread of SARS-CoV-2 in medical facilities as well as in society.We understand that the concept of “immune survivors” needs further discussion. No information is available on how long or the titer of neutralizing antibody required for protection from infection. We have just started to measure antibody levels against SARS-CoV-2 in healthcare workers in our hospital. This project will provide further information in the battle against the SARS-CoV-2 infection. (Clinical trial registration number: UMIN 000039997)

RTS,S/AS01E immunization increases antibody responses to vaccine-unrelated Plasmodium falciparum antigens associated with protection against clinical malaria in African children: a case-control study

BackgroundVaccination and naturally acquired immunity against microbial pathogens may have complex interactions that influence disease outcomes. To date, only vaccine-specific immune responses have routinely been investigated in malaria vaccine trials conducted in endemic areas. We hypothesized that RTS,S/A01E immunization affects acquisition of antibodies to Plasmodium falciparum antigens not included in the vaccine and that such responses have an impact on overall malaria protective immunity.MethodsWe evaluated IgM and IgG responses to 38 P. falciparum proteins putatively involved in naturally acquired immunity to malaria in 195 young children participating in a case-control study nested within the African phase 3 clinical trial of RTS,S/AS01E (MAL055 NCT00866619) in two sites of different transmission intensity (Kintampo high and Manhiça moderate/low). We measured antibody levels by quantitative suspension array technology and applied regression models, multimarker analysis, and machine learning techniques to analyze factors affecting their levels and correlates of protection.ResultsRTS,S/AS01E immunization decreased antibody responses to parasite antigens considered as markers of exposure (MSP142, AMA1) and levels correlated with risk of clinical malaria over 1-year follow-up. In addition, we show for the first time that RTS,S vaccination increased IgG levels to a specific group of pre-erythrocytic and blood-stage antigens (MSP5, MSP1 block 2, RH4.2, EBA140, and SSP2/TRAP) which levels correlated with protection against clinical malaria (odds ratio [95% confidence interval] 0.53 [0.3–0.93], p = 0.03, for MSP1; 0.52 [0.26–0.98], p = 0.05, for SSP2) in multivariable logistic regression analyses.ConclusionsIncreased antibody responses to specific P. falciparum antigens in subjects immunized with this partially efficacious vaccine upon natural infection may contribute to overall protective immunity against malaria. Inclusion of such antigens in multivalent constructs could result in more efficacious second-generation multistage vaccines.

Serological analysis of Ebola virus survivors and close contacts in Sierra Leone: A cross-sectional study.

The 2013–2016 Ebola virus outbreak in West Africa was the largest and deadliest outbreak to date. Here we conducted a serological study to examine the antibody levels in survivors and the seroconversion in close contacts who took care of Ebola-infected individuals, but did not develop symptoms of Ebola virus disease. In March 2017, we collected blood samples from 481 individuals in Makeni, Sierra Leone: 214 survivors and 267 close contacts. Using commercial, quantitative ELISAs, we tested the plasma for IgG-specific antibodies against three major viral antigens: GP, the only viral glycoprotein expressed on the virus surface; NP, the most abundant viral protein; and VP40, a major structural protein of Zaire ebolavirus. We also determined neutralizing antibody titers. In the cohort of Ebola survivors, 97.7% of samples (209/214) had measurable antibody levels against GP, NP, and/or VP40. Of these positive samples, all but one had measurable neutralizing antibody titers against Ebola virus. For the close contacts, up to 12.7% (34/267) may have experienced a subclinical virus infection as indicated by detectable antibodies against GP. Further investigation is warranted to determine whether these close contacts truly experienced subclinical infections and whether these asymptomatic infections played a role in the dynamics of transmission.

Development and Standardization of a High-Throughput Multiplex Immunoassay for the Simultaneous Quantification of Specific Antibodies to Five Respiratory Syncytial Virus Proteins.

Human respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in (premature) newborns and causes respiratory illness in the elderly. Different monoclonal antibody (MAb) and vaccine candidates are in development worldwide and will hopefully become available within the near future. To implement such RSV vaccines, adequate decisions about immunization schedules and the different target group(s) need to be made, for which the assessment of antibody levels against RSV is essential. To survey RSV antigen-specific antibody levels, we developed a serological multiplex immunoassay (MIA) that determines and distinguishes antibodies against the five RSV glycoproteins postfusion F, prefusion F, Ga, Gb, and N simultaneously. The standardized RSV pentaplex MIA is sensitive, highly reproducible, and specific for the five RSV proteins. The preservation of the conformational structure of the immunodominant site Ø of prefusion F after conjugation to the beads has been confirmed. Importantly, good correlation is obtained between the microneutralization test and the MIA for all five proteins, resulting in an arbitrarily chosen cutoff value of prefusion F antibody levels for seropositivity in the microneutralization assay. The wide dynamic range requiring only two serum sample dilutions makes the RSV-MIA a high-throughput assay very suitable for (large-scale) serosurveillance and vaccine clinical studies.IMPORTANCE In view of vaccine and monoclonal development to reduce hospitalization and death due to lower respiratory tract infection caused by RSV, assessment of antibody levels against RSV is essential. This newly developed multiplex immunoassay is able to measure antibody levels against five RSV proteins simultaneously. This can provide valuable insight into the dynamics of (maternal) antibody levels and RSV infection in infants and toddlers during the first few years of life, when primary RSV infection occurs.

Development of a HA1-specific enzyme-linked immunosorbent assay against pandemic influenza virus A H1N1.

PurposeEnzyme-linked immunosorbent assay (ELISA) has been used in the diverse field to evaluate influenza virus infection; for the surveillance, diagnosis, efficacy evaluation, and development of the vaccine. The aim of this study was to establish an ELISA for detecting HA strain-specific antibodies using recombinant pandemic A H1N1 (pH1N1) HA1 (rHA1) protein.Materials and MethodsrHA1 was produced in baculovirus system. The clinical performance of the developed ELISA was validated using human serum samples, by comparison with standard methods for detecting a neutralizing antibody; hemagglutination inhibition (HI) assay and microneutralization test (MNT). The ability of the ELISA system to evaluate the efficacy test of an influenza vaccine was explored by measuring antibody levels in the serum of vaccinated mice.ResultsOur ELISA could detect anti-rHA1 antibody in influenza-infected patients and vaccinated subjects. Compared to HI assay and MNT as reference methods, our method showed good performance in detection of anti-rHA1 antibody. Detection of the anti-rHA1 antibody in vaccinated mice and its correlation with titers in HI assay was also proved in a mice model.ConclusionAn ELISA system using rHA1 of pH1N1 influenza virus was developed, and showed good clinical performance in diagnosis of influenza virus infection and evaluation of the vaccination efficacy in both human and animal models.

Associations of pathogen-specific and host-specific characteristics with disease outcome in patients with Staphylococcus aureus bacteremic pneumonia.

ObjectiveTo understand the relationships of Staphylococcus aureus (SA) bacteremic pneumonia (SABP) outcome with patient‐specific and SA‐specific variables.MethodsWe analysed SA bloodstream isolates and matching sera in SABP patients by sequencing SA isolates (n = 50) and measuring in vitro AT production, haemolytic activity and expression of ClfA and ClfB. Controls were sera from gram‐negative bacteremia patients with or without pneumonia and uninfected subjects. Levels of IgGs, IgMs and neutralizing antibodies (NAbs) against SA antigens were quantified and analysed by one‐way ANOVA. Associations of patient outcomes with patient variables, antibody levels and isolate characteristics were evaluated by univariate and multivariate logistic regression analyses.ResultsSABP patients had higher levels of IgGs against eight virulence factors and anti‐alpha toxin (AT) NAbs than uninfected controls. Levels of IgG against AT and IgMs against ClfA, FnbpA and SdrC were higher in clinically cured SABP patients than in clinical failures. Anti‐LukAB NAb levels were elevated in all cohorts. Increased odds of cure correlated with higher haemolytic activity of SA strains, longer time between surgery and bacteremia (> 30 days), longer duration of antibiotic therapy, lower acute physiology and total APACHE II scores, lack of persistent fever for > 72 h and higher levels of antibodies against AT (IgG), ClfA (IgM), FnbpA (IgM) and SdrC (IgM).DiscussionLimitations included the cross‐sectional observational nature of the study, small sample size and inability to measure antibody levels against all SA virulence factors.ConclusionOur results suggest that SABP patients may benefit from immunotherapy targeting multiple SA antigens.

Protective immune response against Toxoplasma gondii elicited by a novel yeast-based vaccine with microneme protein 16

Toxoplasma gondii is an obligate intracellular protozoan that can invade all eukaryotic cells and infect all warm-blood animals, causing the important zoonosis toxoplasmosis. Invasion of host cells is the key step necessary for T. gondii to complete its life cycle and microneme proteins play an important role in attachment and invasion of host cells. Microneme protein 16 (TgMIC16) is a new protective protein in T. gondii and belongs to transmembrane microneme proteins (TM-MIC). The TM-MICs are released onto the parasite’s surface as complexes capable of interacting with host cell receptors. In the present study, we expressed the TgMIC16 protein on the surface of Saccharomyce cerevisiae (pCTCON2-TgMIC16/EBY100) and evaluated it as a potential vaccine for BALB/c mice against challenge infection with the RH strain of T. gondii. We immunized BALB/c mice both orally and intraperitoneally. After three immunizations, the immune response was evaluated by measuring antibody levels, lymphocyte proliferative responses, percentages of CD4+ and CD8+ T lymphocytes, cytokine production, and the survival times of challenged mice. The results showed that the pCTCON2-TgMIC16/EBY100 vaccine stimulated humoral and cellular immune responses. In addition, mice immunized with the pCTCON2-TgMIC16/EBY100 vaccine showed increased survival times compared with non-immunized controls. In summary, TgMIC16 displayed on the cell surface of S. cerevisiae could be used as potential vaccine against toxoplasmosis.

Methyl-CpG binding domain protein-1 polymorphisms impact immune homeostatic levels of IgG

Abstract Host immunity plays a key role in protection against infection, however, there is significant individual-to-individual variation in the induction and quality of immune response that is driven in part by genetic factors. A growing body of evidence suggests that variation in immune status prior to specific insult may influence subsequent response to vaccination or pathogen challenge. Therefore, understanding the variation in immune status prior to specific insult is necessary for assessing variability in immune response to vaccination or pathogen challenge. The Collaborative Cross (CC) mouse population is a genetically diverse population that was designed to mimic the genetic and phenotypic diversity that is observed in humans and allows us to investigate mechanistically the contribution of genetics to complex phenotypes. Consequently, we asked whether there was variation in antibody levels in 57 CC strains prior to pathogen challenge and if that variation was associated with specific genetic factors. To assess this, we measured antibody levels for IgM, total IgG, IgG1, IgG2a, IgG2b, and IgG2c by ELISA and mapped quantitative trait loci (QTL) associated with variation in antibody concentrations. We observed substantial variation in all antibody isotypes measured. We also identified a suggestive (p=0.1) QTL on chromosome 18. Allele effects show that the C57Bl/6J allele at the locus of interest is linked to higher levels of total IgG. Subsequent analysis of genes under the QTL identified Methyl-CpG binding domain protein-1 (Mbd1) as the most promising candidate gene associated with total IgG variation. Ongoing studies are evaluating the role of Mbd1 in the regulation of total IgG production.

Epitope-Specific Humoral Responses to Human Cytomegalovirus Glycoprotein-B Vaccine With MF59: Anti-AD2 Levels Correlate With Protection From Viremia.

The human cytomegalovirus (HCMV) virion envelope protein glycoprotein B (gB) is essential for viral entry and represents a major target for humoral responses following infection. Previously, a phase 2 placebo-controlled clinical trial conducted in solid organ transplant candidates demonstrated that vaccination with gB plus MF59 adjuvant significantly increased gB enzyme-linked immunosorbent assay (ELISA) antibody levels whose titer correlated directly with protection against posttransplant viremia. The aim of the current study was to investigate in more detail this protective humoral response in vaccinated seropositive transplant recipients. We focused on 4 key antigenic domains (AD) of gB (AD1, AD2, AD4, and AD5), measuring antibody levels in patient sera and correlating these with posttransplant HCMV viremia. Vaccination of seropositive patients significantly boosted preexisting antibody levels against the immunodominant region AD1 as well as against AD2, AD4, and AD5. A decreased incidence of viremia correlated with higher antibody levels against AD2 but not with antibody levels against the other 3 ADs. Overall, these data support the hypothesis that antibodies against AD2 are a major component of the immune protection of seropositives seen following vaccination with gB/MF59 vaccine and identify a correlate of protective immunity in allograft patients.

Is hepatitis B vaccination performed at infant and adolescent age able to provide long-term immunological memory? An observational study on healthcare students and workers in Florence, Italy

ABSTRACTUniversal vaccination programmes against Hepatitis B Virus (HBV) have significantly reduced the burden of the disease; nevertheless, HBV infection remains a relevant issue for high-risk subjects, such as healthcare workers (HCWs), who may potentially be exposed to blood or body fluids. Our study evaluates the long-term duration of the immunological memory of HBV vaccination 11–23 years after primary immunization by examining the response to booster doses in HCWs and students of health disciplines at Careggi Teaching Hospital in Florence (Italy). All participants (n = 2,203) had received a complete HBV immunization course in infancy or adolescence. Blood samples were collected to measure antibody levels against the HBV surface antigen (anti-HBs); an anti-HBs titre <10 mIU/mL was considered as negative. The administration of the vaccination course during infancy induced lower long-term anti-HBs titres compared to those in case of vaccination performed during adolescence (titre <10 mIU/mL: 51.1% and 12.2% respectively; p < 0.001), also considering that an equal number of years has elapsed since vaccination. A booster dose administered to subjects vaccinated in infancy is able to induce anamnestic immunological response in a higher percentage of vaccinated people (p < 0.001). Few subjects (n. = 4) accepted a fifth dose of vaccine in the case of persistent anti-HBs negative titres; this aspect requires further investigation.The total absence of acute hepatitis B among vaccinated subjects suggests that the long incubation period of the disease allows the activation of immunologic memory mechanisms, which is also true in case of low anti-HBs level. In conclusion HCWs still represent a high-risk category; it is therefore, necessary to increase efforts to protect and vaccinate these subjects.

A novel rapid direct haemagglutination-inhibition assay for measurements of humoral immune response against non-haemagglutinating Fowlpox virus strains in vaccinated chickens.

Fowlpox (FP) is a serious disease in chickens caused by Fowlpox virus (FPV). One method currently used to control FPV is vaccination followed by confirmation that antibody titres are protective using the indirect haemagglutination assay (IHA). The direct haemagglutination inhibition (HI) assay is not done because most FPV strains do not agglutinate chicken red blood cells (RBCs). A novel FPV strain TPV-1 which agglutinates chicken RBCs was discovered recently and enabled a direct HI assay to be conducted using homologous sera. This study is therefore aimed at assessing the direct HI assay using a recently discovered novel haemagglutinating FPV strain TPV-1 in chickens vaccinated with a commercial vaccine containing a non-haemagglutinating FPV.Chicks vaccinated with FPV at 1 day-old had antibody geometric mean titres (GMT) of log2 3.7 at 7 days after vaccination and log2 8.0 at 28 days after vaccination when tested in the direct HI. Chickens vaccinated at 6 weeks-old had antibody geometric mean titres (GMT) of log2 5.0 at 7 days after vaccination and log2 8.4 at 28 days after vaccination when tested in the direct HI. The GMT recorded 28 days after vaccination was slightly higher in chickens vaccinated at 6-week-old than in chicks vaccinated at one-day-old. However, this difference was not significant (P > 0.05). All vaccinated chickens showed “takes”. No antibody response to FPV and “takes” were detected in unvaccinated chickens (GMT < 1). There was a slightly higher GMT in chickens of all ages throughout the observation period when the standard assay, the passive (indirect) haemagglutination was used (Overall GMT reached log2 9.3 ±.0.3 on day 28). However, the difference between the two assays was not significant (P > 0.05).ConclusionThese findings indicate that a simple and rapid direct HI assay using the FPV TPV-1 strain as antigen may be used to measure antibody levels in chickens vaccinated with non-haemagglutinating strains of FPV, and that the titres are comparable to those obtained by indirect IHA.