Monitoring SARS-CoV-2 IgA, IgM and IgG antibodies in dried blood and saliva samples using antibody proximity extension assays (AbPEA).

Using a modified proximity extension assay, total and immunoglobulin (Ig) class-specific anti-SARS-CoV-2 antibodies were sensitively and conveniently detected directly from ø1.2 mm discs cut from dried blood and saliva spots (DBS and DSS) without the need for elution. For total Ig detection, antigen probes were prepared by conjugating recombinant spike protein subunit 1 (S1-RBD) to a pair of oligonucleotides. To detect isotype-specific antibody reactivity, one antigen probe was replaced with oligonucleotide-conjugated antibodies specific for antibody isotypes. Binding of pairs of oligonucleotide-conjugated probes to antibodies in patient samples brings oligonucleotides in proximity. An added DNA polymerase uses a transient hybridization between the oligonucleotides to prime synthesis of a DNA strand, which serves as a DNA amplicon that is quantified by real-time PCR. The S1-RBD-specific IgG, IgM, and IgA antibodies in DBS samples collected over the course of a first and second vaccination exhibited kinetics consistent with previous reports. Both DBS and DSS collected from 42 individuals in the autumn of 2023 showed significant level of total S1-RBD antibodies with a correlation of R = 0.70. However, levels in DSS were generally 10 to 100-fold lower than in DBS. Anti-S1-RBD IgG and IgA in DSS demonstrated a correlation of R = 0.6.

Monoclonal antibody development advances immunological research in horses.

Host immune analyses require specific reagents to identify cellular and soluble components of the immune system. These immune reagents are often species-specific. For horses, various immunological tools have been developed and tested by different initiatives during the past decades. This article summarizes the development of well characterized monoclonal antibodies (mAbs) for equine immune cells, immunoglobulin isotypes, cytokines, and chemokines.

B cell clonality in cancer.

Carcinogenesis in the process of long-term co-evolution of tumor cells and immune environment essentially becomes possible due to incorrect decisions made, remembered, and reproduced by the immune system at the level of clonal populations of antigen-specific T- and B-lymphocytes. Tumor-immunity interaction determines the nature of such errors and, consequently, delineates the possible ways of successful immunotherapeutic intervention. It is generally recognized that tumor-infiltrating B cells (TIL-B) can play both pro-tumor and anti-tumor roles. However, the exact mechanisms that determine the contribution of clonal B cell lineages with different specificities and functions remain largely unclear. This is due to the variability of cancer types, the molecular heterogeneity of tumor cells, and, to a large extent, the individual pattern of each immune response. Further progress requires detailed investigation of the functional properties and phenotypes of clonally heterogeneous B cells in relation to their antigenic specificities, which determine the functionality of both effector B lymphocytes and immunoglobulins produced in the tumor environment. Based on a real understanding of the role of clonal antigen-specific populations of B lymphocytes in the tumor microenvironment, we need to learn how to develop new methods of targeted immunotherapy, as well as adapt existing treatment options to the specific needs of different patients and patient subgroups. In this review, we will cover B cells functional diversity and their multifaceted roles in the tumor environment.

Modelling the assembly and flexibility of antibody structures.

Antibodies are large protein assemblies capable of both specifically recognising antigens and engaging with other proteins and receptors to coordinate immune action. Traditionally, structural studies have been dedicated to antibody variable regions, but efforts to determine and model full-length antibody structures are emerging. Here we review the current knowledge on modelling the structures of antibody assemblies, focusing on their conformational flexibility and the challenge this poses to obtaining and evaluating structural models. Integrative modelling approaches, combining experiments (cryo-electron microscopy, mass spectrometry, etc.) and computational methods (molecular dynamics simulations, deep-learning based approaches, etc.), hold the promise to map the complex conformational landscape of full-length antibody structures.

Characterization of SARS-CoV-2 humoral immune response in a subject with unique sampling: A case report.

BACKGROUND: The development of vaccine candidates for COVID-19, and the administration of booster vaccines, has meant a significant reduction in COVID-19 related deaths world-wide and the easing of global restrictions. However, new variants of SARS-CoV-2 have emerged with less susceptibility to vaccine induced immunity leading to breakthrough infections among vaccinated people. It is generally acknowledged that immunoglobulins play the major role in immune-protection, primarily through binding to the SARS-COV-2 receptor binding domain (RBD) and thereby inhibiting viral binding to the ACE2 receptor. However, there are limited investigations of anti-RBD isotypes (IgM, IgG, IgA) and IgG subclasses (IgG1-4) over the course of vaccination and breakthrough infection. METHOD: In this study, SARS-CoV-2 humoral immunity is examined in a single subject with unique longitudinal sampling. Over a two year period, the subject received three doses of vaccine, had two active breakthrough infections and 22 blood samples collected. Serological testing included anti-nucleocapsid total antibodies, anti-RBD total antibodies, IgG, IgA, IgM and IgG subclasses, neutralization and ACE2 inhibition against the wildtype (WT), Delta and Omicron variants. RESULTS: Vaccination and breakthrough infections induced IgG, specifically IgG1 and IgG4 as well as IgM and IgA. IgG1 and IgG4 responses were cross reactive and associated with broad inhibition. CONCLUSION: The findings here provide novel insights into humoral immune response characteristics associated with SARS-CoV-2 breakthrough infections.

Evolution of the Immunoglobulin Isotypes-Variations of Biophysical Properties among Animal Classes.

The adaptive immune system arose around 500 million years ago in jawed fish, and, since then, it has mediated the immune defense against pathogens in all vertebrates. Antibodies play a central role in the immune reaction, recognizing and attacking external invaders. During the evolutionary process, several immunoglobulin isotypes emerged, each having a characteristic structural organization and dedicated function. In this work, we investigate the evolution of the immunoglobulin isotypes, in order to highlight the relevant features that were preserved over time and the parts that, instead, mutated. The residues that are coupled in the evolution process are often involved in intra- or interdomain interactions, meaning that they are fundamental to maintaining the immunoglobulin fold and to ensuring interactions with other domains. The explosive growth of available sequences allows us to point out the evolutionary conserved residues and compare the biophysical properties among different animal classes and isotypes. Our study offers a general overview of the evolution of immunoglobulin isotypes and advances the knowledge of their characteristic biophysical properties, as a first step in guiding protein design from evolution.

Antigen-Specific Antibody Signature Is Associated with COVID-19 Outcome.

Numerous studies have focused on inflammation-related markers to understand COVID-19. In this study, we performed a comparative analysis of spike (S) and nucleocapsid (N) protein-specific IgA, total IgG and IgG subclass response in COVID-19 patients and compared this to their disease outcome. We observed that the SARS-CoV-2 infection elicits a robust IgA and IgG response against the N-terminal (N1) and C-terminal (N3) region of the N protein, whereas we failed to detect IgA antibodies and observed a weak IgG response against the disordered linker region (N2) in COVID-19 patients. N and S protein-specific IgG1, IgG2 and IgG3 response was significantly elevated in hospitalized patients with severe disease compared to outpatients with non-severe disease. IgA and total IgG antibody reactivity gradually increased after the first week of symptoms. Magnitude of RBD-ACE2 blocking antibodies identified in a competitive assay and neutralizing antibodies detected by PRNT assay correlated with disease severity. Generally, the IgA and total IgG response between the discharged and deceased COVID-19 patients was similar. However, significant differences in the ratio of IgG subclass antibodies were observed between discharged and deceased patients, especially towards the disordered linker region of the N protein. Overall, SARS-CoV-2 infection is linked to an elevated blood antibody response in severe patients compared to non-severe patients. Monitoring of antigen-specific serological response could be an important tool to accompany disease progression and improve outcomes.

Comparative Detection of Immunoglobulin Isotypes and Subclasses against Toxoplasma gondii Soluble Antigen in Serum and Colostrum Samples from Puerperal Women.

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite that can infect several species, including humans, and can cause severe damage to the fetus when the infection occurs during pregnancy. The environment and/or food contamination are critical to spreading the infection. Human milk is rich in nutrients and bioactive elements that provide growth and development of the immune system of the newborn. All isotypes of immunoglobulins are present in human colostrum and they are produced from systemic or local sources. Breastfeeding protects the infant against various pathogens, but there is no conclusive study to detect IgG subclasses in colostrum against T. gondii. Therefore, the aim of this study was to detect and evaluate the presence of antibody isotypes against T. gondii in paired samples of serum and colostrum. METHODS: The study included 283 puerperal patients. ELISA (Enzyme-Linked Immunosorbent Assay) for detection of anti-T. gondii-specific IgM, IgA, and IgG isotypes and IgG1, IgG3, and IgG4 subclasses were conducted on paired samples of serum and colostrum. RESULTS: It was found that 45.9%, 6.0%, and 2.1% of serum samples and 45.2%, 7.1%, and 2.1% of colostrum samples were positive for IgG, IgM, and IgA, respectively. Specific IgG1, IgG3, and IgG4 were positive, respectively, in 98.5%, 54.6%, and 44.6% of serum samples, in contrast with 56.9%, 78.5%, and 34.6% of colostrum samples. Thus, the predominant reactivity of IgG subclasses against T. gondii was IgG1 in serum and IgG3 in colostrum. The higher percentage of positive samples and higher levels of anti-T. gondii IgG3 antibodies were observed in colostrum, when compared to serum samples, suggesting a local production of this subclass. IgG3 and IgG1 subclasses presented different percentages of positivity in serum and colostrum. Only the IgG1 subclass showed a significant correlation between the levels of anti-T. gondii in serum and colostrum, suggesting that IgG1 in breast milk comes from a systemic source. IgG4 showed a similar percentage of positivity in both sample types, but no significant correlation was observed between their levels. CONCLUSION: Colostrum presents representative levels of IgM, IgA, IgG1, IgG3, and IgG4 antibodies specific to T. gondii. The detection of these antibodies presents the potential for diagnostic application of colostrum samples to better identify the diagnostic status of T. gondii infection, especially during the acute phase. In addition, breastfeeding can also be a possible source of protective antibodies for the newborn against toxoplasmosis, an anthropozoonosis maintained by environmental infection, which interferes in the public health of many countries.

Immune cell-antibody interactions in health and disease.

The human immune system safeguards against pathogens through a multitude of cellular and molecular signals, involving different components of the innate and adaptive response. Contrastingly, autoimmune diseases, allergic conditions, and cancer evoke different aspects of these otherwise protective processes. Understanding the immunological hallmarks for each pathological setting is essential for improving prevention, diagnosis, prognosis, and treatment. The activatory states of immune effector cells, especially in relation to their direct or indirect interactions with antibodies, are important determinants of an efficient, protective response that results in target clearance and improved clinical outcomes. Dysregulation of effector cells and their functions alongside alternatively activated humoral immune responses may contribute to several chronic diseases including allergic inflammation, autoimmune disorders and cancer. This Review Series brings to the forefront several key activation and regulatory features of immune effector cells in different diseases including cancer, infection allergy, and autoimmunity. Specific attention is drawn on how antibodies can impact effector cell states, and their pro-inflammatory and immune protective functions. Articles in this Series discuss different effector cells and antibody isotypes in infection, inflammation, tolerance and cancer immune surveillance, covering basic and translational mechanisms, clinical and epidemiological insights into these immune responses. Understanding the critical attributes of immune cells, especially those needed to effectively engage antibodies, will undoubtedly help better exploit their potential for disease management and therapy.

B-Cell Homeostasis Is Maintained During Two Months of Head-Down Tilt Bed Rest With or Without Antioxidant Supplementation.

Alterations of the immune system could seriously impair the ability to combat infections during future long-duration space missions. However, little is known about the effects of spaceflight on the B-cell compartment. Given the limited access to astronaut samples, we addressed this question using blood samples collected from 20 healthy male volunteers subjected to long-duration bed rest, an Earth-based analog of spaceflight. Hematopoietic progenitors, white blood cells, total lymphocytes and B-cells, four B-cell subsets, immunoglobulin isotypes, six cytokines involved in inflammation, cortisone and cortisol were quantified at five time points. Tibia microarchitecture was also studied. Moreover, we investigated the efficiency of antioxidant supplementation with a cocktail including polyphenols, omega 3, vitamin E and selenium. Our results show that circulating hematopoietic progenitors, white blood cells, total lymphocytes and B-cells, and B-cell subsets were not affected by bed rest. Cytokine quantification suggested a lower systemic inflammatory status, supported by an increase in serum cortisone, during bed rest. These data confirm the in vivo hormonal dysregulation of immunity observed in astronauts and show that bed rest does not alter B-cell homeostasis. This lack of an impact of long-term bed rest on B-cell homeostasis can, at least partially, be explained by limited bone remodeling. None of the evaluated parameters were affected by the administration of the antioxidant supplement. The non-effectiveness of the supplement may be because the diet provided to the non-supplemented and supplemented volunteers already contained sufficient antioxidants. Given the limitations of this model, further studies will be required to determine whether B-cell homeostasis is affected, especially during future deep-space exploration missions that will be of unprecedented durations.

High throughput surface plasmon resonance imaging method for clinical detection of presence and strength of binding of IgM, IgG and IgA antibodies against SARS-CoV-2 during CoViD-19 infection.

Surface Plasmon Resonance imaging (SPRi) was used to determine the presence and strength of binding of IgG, IgM and IgA against the Receptor Binding Domain (RBD) of SARS-CoV-2 in sera of 102 CoViD-19 and non-CoViD-19 patients. The SPRi assay simultaneously measures the antibody isotype levels and the strength of binding to the RBD of ultimate 384 patient samples in one run. It turns out that during the course of the disease, the IgG levels and strength of binding increased while generally the IgM and IgA levels go down. Recovered patients all show high strength of binding of the IgG type to the RBD protein. The anti-RBD immunoglobulins SPRi assay provides additional insights in the immune status of patients recovering from CoViD-19. This new high throughput method can be applied for the assessment of the quality of the immune reaction of healthy individuals to SARS-CoV-2 and its mutants in vaccination programs.•Surface Plasmon Resonance imaging is an unprecedented technology for high throughput screening of antibody profiling of CoViD19 patients.•Fingerprinting of isotypes IgM, IgG and IgA can be performed for 384 patients in one run.•An affinity maturation effect was shown for patients recovering from CoViD19.

Detection of Antibody Responses against SARS-CoV-2 in Plasma and Saliva from Vaccinated and Infected Individuals.

Antibodies (Abs) are essential for the host immune response against SARS-CoV-2, and all the vaccines developed so far have been designed to induce Abs targeting the SARS-CoV-2 spike. Many studies have examined Ab responses in the blood from vaccinated and infected individuals. However, since SARS-CoV-2 is a respiratory virus, it is also critical to understand the mucosal Ab responses at the sites of initial virus exposure. Here, we examined plasma versus saliva Ab responses in vaccinated and convalescent patients. Although saliva levels were significantly lower, a strong correlation was observed between plasma and saliva total Ig levels against all SARS-CoV-2 antigens tested. Virus-specific IgG1 responses predominated in both saliva and plasma, while a lower prevalence of IgM and IgA1 Abs was observed in saliva. Antiviral activities of plasma Abs were also studied. Neutralization titers against the initial WA1 (D614G), B.1.1.7 (alpha) and B.1.617.2 (delta) strains were similar but lower against the B.1.351 (beta) strain. Spike-specific antibody-dependent cellular phagocytosis (ADCP) activities were also detected and the levels correlated with spike-binding Ig titers. Interestingly, while neutralization and ADCP potencies of vaccinated and convalescent groups were comparable, enhanced complement deposition to spike-specific Abs was noted in vaccinated versus convalescent groups and corresponded with higher levels of IgG1 plus IgG3 among the vaccinated individuals. Altogether, this study demonstrates the detection of Ab responses after vaccination or infection in plasma and saliva that correlate significantly, although Ig isotypic differences were noted. The induced plasma Abs displayed Fab-mediated and Fc-dependent functions with comparable neutralization and ADCP potencies, but a greater capacity to activate complement was elicited upon vaccination.

Detection of Antibody Responses Against SARS-CoV-2 in Plasma and Saliva From Vaccinated and Infected Individuals.

Antibodies (Abs) are essential for the host immune response against SARS-CoV-2, and all the vaccines developed so far have been designed to induce Abs targeting the SARS-CoV-2 spike. Many studies have examined Ab responses in the blood from vaccinated and infected individuals. However, since SARS-CoV-2 is a respiratory virus, it is also critical to understand the mucosal Ab responses at the sites of initial virus exposure. Here, we examined plasma versus saliva Ab responses in vaccinated and convalescent patients. Although saliva levels were significantly lower, a strong correlation was observed between plasma and saliva total Ig levels against all SARS-CoV-2 antigens tested. Virus-specific IgG1 responses predominated in both saliva and plasma, while a lower prevalence of IgM and IgA1 Abs was observed in saliva. Antiviral activities of plasma Abs were also studied. Neutralization titers against the initial WA1 (D614G), B.1.1.7 (alpha) and B.1.617.2 (delta) strains were similar but lower against the B.1.351 (beta) strain. Spike-specific antibody-dependent cellular phagocytosis (ADCP) activities were also detected and the levels correlated with spike-binding Ig titers. Interestingly, while neutralization and ADCP potencies of vaccinated and convalescent groups were comparable, enhanced complement deposition to spike-specific Abs was noted in vaccinated versus convalescent groups and corresponded with higher levels of IgG1 plus IgG3 among the vaccinated individuals. Altogether, this study demonstrates the detection of Ab responses after vaccination or infection in plasma and saliva that correlate significantly, although Ig isotypic differences were noted. The induced plasma Abs displayed Fab-mediated and Fc-dependent functions with comparable neutralization and ADCP potencies, but a greater capacity to activate complement was elicited upon vaccination.

Role of Immunoglobulin M and A Antibodies in the Neutralization of Severe Acute Respiratory Syndrome Coronavirus 2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people globally. Virus infection requires the receptor-binding domain (RBD) of the spike protein. Although studies have demonstrated anti-spike and -RBD antibodies to be protective in animal models, and convalescent plasma as a promising therapeutic option, little is known about immunoglobulin isotypes capable of blocking infection. METHODS: We studied spike- and RBD-specific immunoglobulin isotypes in convalescent and acute plasma/serum samples using a multiplex bead assay. We also determined virus neutralization activities in plasma and serum samples, and purified immunoglobulin fractions using a vesicular stomatitis pseudovirus assay. RESULTS: Spike- and RBD-specific immunoglobulin (Ig) M, IgG1, and IgA1 were produced by all or nearly all subjects at variable levels and detected early after infection. All samples displayed neutralizing activity. Regression analyses revealed that IgM and IgG1 contributed most to neutralization, consistent with IgM and IgG fractions' neutralization potency. IgA also exhibited neutralizing activity, but with lower potency. CONCLUSION: IgG, IgM, and IgA are critical components of convalescent plasma used for treatment of coronavirus disease 2019 (COVID-19).

Role of IgM and IgA Antibodies in the Neutralization of SARS-CoV-2.

BACKGROUND: SARS-CoV-2 has infected millions of people globally. Virus infection requires the receptor-binding domain (RBD) of the spike protein. Although studies have demonstrated anti-spike and - RBD antibodies to be protective in animal models, and convalescent plasma as a promising therapeutic option, little is known about immunoglobulin (Ig) isotypes capable of blocking infection. METHODS: We studied spike- and RBD-specific Ig isotypes in convalescent and acute plasma/sera using a multiplex bead assay. We also determined virus neutralization activities in plasma, sera, and purified Ig fractions using a VSV pseudovirus assay. RESULTS: Spike- and RBD-specific IgM, IgG1, and IgA1 were produced by all or nearly all subjects at variable levels and detected early after infection. All samples displayed neutralizing activity. Regression analyses revealed that IgM and IgG1 contributed most to neutralization, consistent with IgM and IgG fractions' neutralization potency. IgA also exhibited neutralizing activity, but with lower potency. CONCLUSION: IgG, IgM and IgA are critical components of convalescent plasma used for COVID-19 treatment.

Envelope-Specific IgG3 and IgG1 Responses Are Associated with Clearance of Acute Hepatitis C Virus Infection.

Hepatitis C virus (HCV) can be cleared naturally in a subset of individuals. However, the asymptomatic nature of acute HCV infection makes the study of the early immune response and defining the correlates of protection challenging. Despite this, there is now strong evidence implicating the humoral immune response, specifically neutralising antibodies, in determining the clearance or chronicity outcomes of primary HCV infection. In general, immunoglobulin G (IgG) plays the major role in viral neutralisation. However, there are limited investigations of anti-HCV envelope protein 2 (E2) isotypes (IgM, IgG, IgA) and IgG subclasses (IgG1-4) in early HCV infection. In this study, using a rare cohort of 14 very recently HCV-infected individuals (4-45 days) with varying disease outcome (n = 7 clearers), the timing and potency of anti-HCV E2 isotypes and IgG subclasses were examined longitudinally, in relation to neutralising antibody activity. Clearance was associated with anti-E2 IgG, specifically IgG1 and IgG3, and appeared essential to prevent the emergence of new HCV variants and the chronic infection outcome. Interestingly, these IgG responses were accompanied by IgM antibodies and were associated with neutralising antibody activity in the subjects who cleared infection. These findings provide novel insights into the early humoral immune response characteristics associated with HCV disease outcome.

Fabrication of Microarrays for the Analysis of Serological Antibody Isotypes against Food Antigens.

Food intolerance is delayed adverse food reactions which follow consumption of specific foods. The underlying mechanisms are not well understood, but food intolerance is often considered as a type 2 hypersensitivity reaction mediated by immunoglobulin G (IgG) antibody. To understand the causes of food intolerance, it is important to investigate sensitization patterns of food-specific IgGs (sIgG) in relation to dietary patterns and physical conditions. Conventional approaches to measure serological IgGs often require large volumes of serum, thus are not suitable for highly multiplexed assays. To overcome this impracticality, we developed a highly sensitive method to screen the sIgGs and other antibody isotypes against 66 antigens with minimal amount of serums. We prepared a microarray by immobilizing food antigens on activated glass slides. Human sera and their dietary information were obtained from 30 subjects. Aliquots (200 nl) of sera were analyzed against 66 food antigens in parallel. sIgG levels were determined and analyzed in relation to subjects' dietary patterns. The levels of antibody isotypes were also examined to understand the relationship between allergy and food intolerance. The developed microarray showed exceptional performances in antibody screening and demonstrated the potential to be used as an automated assay system.

Circulating innate and adaptive immunity against anti-Haemophilus influenzae type b.

Haemophilus influenzae type b (Hib) are one of most dangerous microbes that occupies the paediatric nasopharyngeal as a commensal opportunistic bacterium, which may lead to meningitis in uncontrolled infection. Colonisation of pharyngeal tissues is the starting point for most H. influenzae infections, which may develop into invasive diseases, such meningitis. The vaccination against Hib in specific, as well as against most of vaccines preventable diseases; in general, play a major role in reducing children (< 5 years old) Hib meningitis from 57/100,000 to the lowest known Hib meningitis incidents in the history. First invented Hib vaccine was licensed in 1985 and contained Hib capsular polysaccharide (CPS); afterward, conjugate vaccines have been innovated and licensed on the road to improve Hib vaccine efficacy. Polyribosylribitol phosphate (PRP) is the main vaccine unite structure. Since anti-CPS antibodies in the serum reflect the extent of the acquired immunity against Hib infections, the concentration of ⩾ 0.15 g/ml of anti-CPS is believed to be an indicator for short-term protection from invasive Hib diseases, whereas one-month post-completion of primary Hib immunization concentration of ⩾ 1.0 g/ml is trusted to be immunological protective. As considered that serum anti-CPS antibodies are effectively linked to protection, the evaluation of antibodies concentration and reconsideration of published worldwide populations antibodies concentration are consider vital strides on the way to accurate valuation of Hib immunity that induced by vaccination; either direct or herd. As documented, some populations; worldwide, still susceptible to invasive Hib infections. Several populations worldwide remain vulnerable to Hib-related infections. We believe that up-to-date review article regarding circulated Hib immunology, represented in anti-Hib antibodies and worldwide Hib incidences will provide a precious information for microbiologists, public health officials, epidemiologists, immunologists, and strategic preventive healthcare executives.

Will Attention by Vaccine Developers to the Host's Nuclear Hormone Levels and Immunocompetence Improve Vaccine Success?

Despite extraordinary advances in fields of immunology and infectious diseases, vaccine development remains a challenge. The development of a respiratory syncytial virus vaccine, for example, has spanned more than 50 years of research with studies of more than 100 vaccine candidates. Dozens of attractive vaccine products have entered clinical trials, but none have completed the path to licensing. Human immunodeficiency virus vaccine development has proven equally difficult, as there is no licensed product after more than 30 years of pre-clinical and clinical research. Here, we examine vaccine development with attention to the host. We discuss how nuclear hormones, including vitamins and sex hormones, can influence responses to vaccines. We show how nuclear hormones interact with regulatory elements of immunoglobulin gene loci and how the deletion of estrogen response elements from gene enhancers will alter patterns of antibody isotype expression. Based on these findings, and findings that nuclear hormone levels are often insufficient or deficient among individuals in both developed and developing countries, we suggest that failed vaccine studies may in some cases reflect weaknesses of the host rather than the product. We encourage analyses of nuclear hormone levels and immunocompetence among study participants in clinical trials to ensure the success of future vaccine programs.

Mixed Chlamydia trachomatis Peptide Antigens Provide a Specific and Sensitive Single-Well Colorimetric Enzyme-Linked Immunosorbent Assay for Detection of Human Anti-C. trachomatis Antibodies.

Sensitive and specific detection of anti-Chlamydia trachomatis antibodies in standard enzyme-linked immunosorbent assays (ELISAs) is compromised by cross-reactivity and poor sensitivity of classical C.trachomatis antigens. Previously, we discovered 48 strongly reactive peptide antigens of C. trachomatis-specific B-cell epitopes from 21 immunodominant proteins. By comprehensive individual testing of 11 top-ranked peptide antigens, we found very high sensitivity and specificity for detection of anti-C. trachomatis antibodies in chemiluminescent ELISAs. The current study established a labor-saving colorimetric ELISA by using a mixture of 12 strongly reactive C. trachomatis peptide antigens (Ctr Mix1) in a single well/serum rather than assaying reactivity to each individual peptide. For performance evaluation, we used a simulated population of 212 anti-C. trachomatis antibody-positive and -negative sera from 125 women with NAAT-confirmed active C. trachomatis infection and from 87 healthy women at low risk for C. trachomatis infection. In comparison to a composite reference standard (CRS) for anti-C. trachomatis antibody status, the Ctr Mix1 IgG ELISA achieved 93.9% sensitivity, significantly superior to the 49% to 79% sensitivities of four commercial anti-C. trachomatis IgG ELISAs, and 98% specificity of all tested assays. Compared to the labor-intensive individual peptide testing, this mixed peptide ELISA retained high specificity with only marginal, ∼5% sensitivity loss. By ROC-AUC, likelihood ratio, and predictive value analyses, the Ctr Mix1 ELISA performed satisfactorily at 10% to 75% prevalence range of anti-C. trachomatis antibodies but significantly better than commercial ELISAs. Thus, the labor-saving mixed peptide colorimetric ELISA format provides simultaneously high specificity and sensitivity for detection of anti-C. trachomatis antibodies.IMPORTANCE For detection of anti-C. trachomatis antibodies by serological assays, use of classical chlamydial antigens results in high cross-reactivity and poor sensitivity. Previously, we discovered 48 strongly reactive peptide antigens of C. trachomatis-specific B-cell epitopes from 21 immunodominant proteins, and individual testing and combined scoring of 5 to 11 peptide antigens provided highly sensitive and specific detection of anti-C. trachomatis antibodies in chemiluminescent ELISAs. To simplify this method, this study established a single-well labor-saving colorimetric ELISA using a mixture of 12 strongly reactive C. trachomatis peptide antigens (Ctr Mix1) for detection of anti-C. trachomatis antibodies. This Ctr Mix1 ELISA (94% sensitivity and 98% specificity) outperformed 4 commercial ELISAs (49% to 79% sensitivity and 98% specificity). This ELISA can be easily implemented and commercialized, with convenient setup for use in nonspecialized laboratories. Thus, this mixed peptide assay with superior specificity and sensitivity will improve serodiagnosis of C. trachomatis infections.