Comparison of Antibody Repertoires against Staphylococcus aureus in Healthy Individuals and in Acutely Infected Patients

The management of staphylococcal diseases is increasingly difficult with present medical approaches. Preventive and therapeutic vaccination is considered to be a promising alternative; however, little is known about immune correlates of protection and disease susceptibility. To better understand the immune recognition of Staphylococcus aureus by the human host, we studied the antistaphylococcal humoral responses in healthy people in comparison to those of patients with invasive diseases. In a series of enzyme-linked immunosorbent assay analyses performed using 19 recombinant staphylococcal cell surface and secreted proteins, we measured a wide range of antibody levels, finding a pronounced heterogeneity among individuals in both donor groups. The analysis revealed marked differences in the antibody repertoires of healthy individuals with or without S. aureus carriage, as well as in those of patients in the acute phase of infection. Most importantly, we identified antigenic proteins for which specific antibodies were missing or underrepresented in infected patients. In contrast to the well-described transient nature of disease-induced antistaphylococcal immune response, it was demonstrated that high-titer antistaphylococcal antibodies are stable for years in healthy individuals. In addition, we provide evidence obtained on the basis of opsonophagocytic and neutralizing activity in vitro assays that circulating antistaphylococcal serum antibodies in healthy donors are functional. In light of these data we suggest that proper serological analysis comparing the preexisting antibody repertoires of hospitalized patients with different outcomes for nosocomial staphylococcal infections could be extremely useful for the evaluation of candidate vaccine antigens in addition to protection data generated with animal models.     

Reconstitution of self-reactive antibody repertoires of autologous plasma IgM in patients with non-Hodgkin's lymphoma following myeloablative therapy

In healthy individuals, natural self-reactive antibody repertoires are restricted to a limited subset of autoantigens that is selected early in development and that remains invariant between individuals through aging. In the present study, we addressed the question of whether self-reactive antibody repertoires of plasma IgM change during high-dose chemotherapy (HDCT) with autologous blood stem cell support and whether antibody repertoires generated during immune reconstitution are similar to those present under physiological conditions. We followed the development of antibody repertoires in patients undergoing HDCT for the treatment of B-cell non-Hodgkin's lymphoma (NHL). Antibody repertoires were investigated by quantitative immunoblotting on whole tissue extracts as sources of self-antigens and by multiparametric statistical analysis of the data. We demonstrate that self-reactive antibody repertoires of plasma IgM of NHL patients prior to HDCT differ from those of healthy individuals, that they change during recovery of immune functions, and that antibody repertoires similar to those of healthy individuals are generated during immune reconstitution. We conclude that the mechanisms responsible for the selection of self-reactive repertoires of autologous plasma IgM during immune reconstitution after HDCT may follow those present under physiological conditions and that immune reconstitution may include a shift from altered toward normal patterns of self-reactivity.     

Immunoglobulin E antibodies against Staphylococcus aureus cell walls in the sera of patients with hyperimmunoglobulinemia E and recurrent staphylococcal infection.

The specificity of antistaphylococcal antibodies of the IgE class in five patients with hyperimmunoglobulinemia E and recurrent staphylococcal infection has been investigated. Purified cell walls were prepared from various staphylococcal strains, and serum immunoglobulin E binding was measured by using a solid-phase radioimmunoassay. Immunoglobulin E binding occurred only with cell walls from Staphylococcus aureus strains, including walls from a teichoic acid-deficient mutant. Immunoglobulin E did not bind to cell wall preparations from the coagulase-negative species S. capitis, S. sciuri subsp. lentus, S. simulans, S. xylosus, staphylococcal strains RB-11 and Armour, and from a group A streptococcus strain CS44. Since the glycan backbone and the tetrapeptide (pentapeptide) subunit of the peptidoglycan of all staphylococcal strains tested are believed to be identical, it is suggested that IgE binding is related to either the peptidoglycan interpeptide bridge or an unknown antigenic structure within the cell wall of S. aureus. The pathophysiological significance of antistaphylococcal immunoglobulin E antibodies in the disorder studied is at present unknown. The formation of immunoglobulin E antibodies to S. aureus cell wall components may be a manifestation of an aberrant immunological response to S. aureus related to the undue susceptibility to staphylococcal infections in these patients.     

Occurrence of antibodies to teichoic acid in patients with diseases other than staphylococcal infection

To determine the usefulness of the teichoic acid antibody (TAA) test in conditions where unspecific viral and bacterial antibodies are often encountered, we measured TAA by the gel-diffusion method in 475 patients without known staphylococcal disease; they included 213 patients with arthritis, 108 with liver diseases, 100 with gastro-intestinal disorders and 54 with acute pharyngitis. Positive controls were 104 patients with Staphylococcus aureus bacteraemia and 203 healthy adults were negative controls. Thirteen (6%) of the healthy adults had positive TAA titres (greater than or equal to 4), and the highest titre was 8 in two people (1%). Positive titres were found in 38% of patients with S. aureus bacteraemia and high titres (greater than or equal to 8) were seen in 24%. Among the patients with arthritis, positive TAA titres were found significantly more often than in healthy controls in patients with Yersinia arthritis (p less than 0.01) and systemic lupus erythematosus (SLE; p less than 0.02). In other patient groups, the percentage of positive TAA titres did not differ significantly from that in healthy adults. Eight (2%) of the 475 patients without known staphylococcal infection had TAA titres greater than or equal to 8 but these high titres were not associated with any particular disease group. Only two of these eight patients had slightly raised antibody to staphylococcal alpha-haemolysin. We conclude that the TAA test cannot be used as a reliable indicator of septic staphylococcal disease in patients with Yersinia arthritis or SLE, but that in general, TAA titres greater than or equal to 8 point strongly to S. aureus infection even in patients with autoimmune or liver diseases.     

Staphylococcus aureus capsular types and antibody response to lung infection in patients with cystic fibrosis.

Chronic respiratory tract infections caused by Staphylococcus aureus are common in patients with cystic fibrosis (CF). Recently, it was shown in a few CF patients that S. aureus isolates produce capsular polysaccharides (CPs). However, it is not known whether this is a common feature and whether an immune response to CPs in CF is detectable. Therefore, we examined 170 S. aureus isolates from CF patients and healthy individuals for production of CP types 5 and 8 by using monoclonal antibodies. We found that CP-producing staphylococcal isolates were randomly distributed among CF patients and healthy carriers. Eighty-five percent of all isolates produced CPs, 77% of which were type 8. Examination of one sputum sample by an immunofluorescence technique suggested that production of CPs is not an in vitro phenomenon. S. aureus isolates from various sites of a single person often yielded more than one CP type. A random distribution of S. aureus strains with CP type 5 or 8 from the skin and respiratory tracts of patients and from the skin of healthy individuals was found. Antibody response to CP types 5 and 8, measured by enzyme-linked immunosorbent assay, was not elevated in CF patients with chronic S. aureus lung infection in comparison with healthy carriers. On the contrary, in CF patients the ratios of antibodies to CP 8 were significantly lower (P less than 0.005; alpha = 0.025). The ratios of antibodies to CP types did not change when monitored longitudinally over several months. This study suggests that the production of CPs is a universal property of S. aureus and that infected CF patients do not have elevated ratios of antibodies to these antigens.     

Antibody responses in patients with staphylococcal septicemia against two Staphylococcus aureus fibrinogen binding proteins: clumping factor and an extracellular fibrinogen binding protein

We analyzed the serum antibody responses against two Staphylococcus aureus fibrinogen binding proteins, the cell-bound clumping factor (Clf) and an extracellular fibrinogen binding protein (Efb). The material consisted of 105 consecutive serum samples from 41 patients suffering from S. aureus septicemia and 72 serum samples from healthy individuals. An enzyme-linked immunosorbent assay (ELISA) was developed. Healthy individuals showed variable levels of antibodies against the studied antigens, and cutoff levels (upper 95th percentile) against these antigens were determined. No correlation was seen between serum antibody levels against Clf and Efb. In acute-phase samples 27% of patients showed positive antibody levels against Clf and 10% showed positive levels against Efb, while in convalescent-phase samples 63% (26 of 41) showed a positive serology against Clf and 49% (20 of 41) showed a positive serology against Efb. Antibody levels against Efb were significantly lower in the acute-phase sera than in sera from healthy individuals (P = 0. 002). An antibody response against Clf was most frequent in patients suffering from osteitis plus septic arthritis and from endocarditis (80% positive). The antibody response against Efb appeared to develop later in the course of disease. A possible biological effect of measured antibodies was demonstrated with the help of an inhibition ELISA, in which both high-titer and low-titer sera inhibited the binding of bacteria to fibrinogen. In conclusion, we have demonstrated in vivo production of S. aureus fibrinogen binding proteins during deep S. aureus infections and a possible diagnostic and prophylactic role of the corresponding serum antibodies in such infections.     

Serum antibodies against Panton–Valentine leukocidin in a normal population and during Staphylococcus aureus infection

To determine whether Staphylococcus aureus Panton–Valentine leukocidin (PVL) is expressed during human infection, anti-PVL antibody titres were compared in patients with PVL-positive and PVL-negative staphylococcal infections, and in patients with no evidence of S. aureus infection. Patients with PVL-positive strains had higher levels of anti-PVL antibodies than individuals of both control groups. The median anti-PVL titre increased 8.6-fold during the course of PVL-positive infection and 1.4-fold during PVL-negative infection. These results indicate that only PVL-positive S. aureus strains elicit significant anti-PVL antibody production in humans, and demonstrate the production of PVL during PVL-positive S. aureus infection. The protective role of this immune response remains to be established.     

Western blot analysis of staphylococcal antibodies present in human sera during health and disease

IgG antibodies directed against Staphylococcus aureus were examined by Western blotting in sera from 15 healthy individuals isolated for a year in Antarctica. Sera reacted with many staphylococcal antigens in whole-cell extracts and individuals showed unique and unchanging blot profiles. The IgG and IgM profiles of patients with deep-seated staphylococcal infections were also examined by Western blotting. Anti-staphylococcal IgM antibodies that reacted with an antigen of apparent molecular mass 31 X 10(3) were present in all patients with staphylococcal disease, and were absent from, or detected in much smaller amounts in, control sera.     

The inverse correlation between Staphylococcus aureus and Streptococcus pneumoniae colonization in infants is not explained by differences in serum antibody levels in the Generation R Study

Colonization rates of Streptococcus pneumoniae and Staphylococcus aureus are inversely correlated in infants. Several studies have searched for determinants of this negative association. We studied the association between antipneumococcal antibodies with Staphylococcus aureus colonization and the association between antistaphylococcal antibodies with pneumococcal colonization in healthy children in the pneumococcal vaccine era. In the first year of life, no association between maternal IgG levels and colonization was seen. In addition, no association between the IgG and IgA levels in the child versus colonization status was seen.     

Nasopharyngeal colonization elicits antibody responses to staphylococcal and pneumococcal proteins that are not associated with a reduced risk of subsequent carriage

Knowledge of the immunological correlates of Staphylococcus aureus and Streptococcus pneumoniae colonization is required for the search for future protein vaccines. We evaluated natural antibody levels against pneumococcal and staphylococcal proteins in relation to previous bacterial colonization with both pathogens. In a randomized controlled trial, nasopharyngeal samples were obtained from children at 1.5, 6, 12, 18, and 24 months and cultured for S. aureus and S. pneumoniae. Approximately 50% of the children were PCV7 vaccinated. Serum IgG against 18 pneumococcal and 40 staphylococcal proteins was semiquantified by Luminex technology from 111 12 month olds and 158 24 month olds. Previous culture-proven S. aureus colonization was associated with higher IgG levels against 6/40 staphylococcal proteins (ClfB, ClfA, Efb, CHIPS, LukD, and LukF [P ≤ 0.001]) compared to noncarriers. Previous pneumococcal colonization was associated with increased IgG levels against 12/18 pneumococcal proteins compared to noncarriers (P ≤ 0.003). Increasing age was associated with higher levels of antibodies to most pneumococcal proteins and lower levels of antibodies to over half the staphylococcal proteins, reflecting natural colonization dynamics. Anti-S. pneumoniae and anti-S. aureus protein antibodies at the age of 12 months were not negatively correlated with subsequent colonization with the homologous species in the following year and did not differ between PCV7-vaccinated and nonvaccinated children. Colonization with S. aureus and S. pneumoniae induces serum IgG against many proteins, predominantly proteins with immune-modulating functions, irrespective of PCV7 vaccination. None of them appeared to be protective against new acquisition with both pathogens, possibly due to the polymorphic nature of those proteins in the circulating bacterial population.     

Instability of Natural Antibody Repertoires in Systemic Lupus Erythematosus Patients, Revealed by Multiparametric Analysis of Serum Antibody Reactivities

Recent views on autoimmune diseases invoke generalized but specific perturbations in antibody repertoires, rather than the clonally restricted or non-specific polyclonal alterations proposed thus far. The present experiments analyse serum antibody reactivities in 24 systemic lupus erythematosus (SLE) patients and 17 healthy controls, using a method that quantitatively scores a large number of antibody reactivities and allows for multiparametric statistical analyses. The results show global but relatively specific perturbations in SLE antibody repertoires, and identify novel disease-associated reactivity patterns. Furthermore, a time series analysis of serum antibodies over 3 months demonstrates instability of natural antibody repertoires in individual SLE patients, contrasting with their remarkable conservation in healthy donors. Moreover, the method used clusters controls and patients independently, and might prove of diagnostic value, once large data bases are established.     

Alterations of self-reactive antibody repertoires in HIV disease: an insight into the role of T cells in the selection of autoreactive B cells

Infection with human immunodeficiency virus (HIV) is characterized by a progressive depletion of CD4(+) T cells that parallels a dysfunction of the B cell compartment and a disturbed recognition of self-antigens. The relationship between T lymphocyte homeostasis and abnormalities in the selection of self-reactive B cells is not clear as yet. We have therefore compared repertoires of natural antibodies of healthy donors and of patients at various stages of HIV infection. The reactivity of IgM and IgG antibodies in plasma of healthy blood donors and of HIV-positive patients with high and low CD4(+) T cell counts was assessed by semi-quantitative immunoblotting using self-antigens extracted from normal human tissues. Repertoires of reactivites were compared between groups of individuals by means of multiparametric statistical analysis. We observed that repertoires of self-reactive IgM and IgG from HIV-seropositive patients exhibited significantly altered patterns of reactivity, as compared to those of healthy controls. Further, self-reactive repertoires of IgM and IgG of patients with high CD4(+) T cell counts differed significantly from those of patients with low CD4(+) T cell counts. A longitudinal analysis of self-reactive antibody repertoires of progressor and non-progressor patients suggested an influence of CD4(+) T cell counts on immunoglobulin reactivity toward self-antigens. These observations support the hypothesis that altered T cell/B cell interactions due to altered CD4(+) T cell help severely impact on the selection of self-reactive antibody repertoires and may contribute to the onset of pathological autoimmunity in HIV disease.     

The response of human B cells to Mycobacterium leprae. Identification of target antigens following polyclonal activation in vitro

We have investigated the B cell response to Mycobacterium leprae in leprosy patients and healthy controls. A comparison of Western-blotted proteins separated by two-dimensional gel electrophoresis and probed with pooled sera from LL and BT patients revealed distinct antigen recognition patterns for the two classifications of the disease. To characterize the circulating B cells capable of producing anti-M. leprae antibodies in vitro, peripheral blood lymphocyte cultures were activated polyclonally with an anti-CD3 mAb. The resulting culture supernatants were used to probe Western-blotted M.leprae proteins and contained antibody reactive with a 10 kd M.leprae antigen. This antibody was absent in stimulated culture supernatants from healthy occupational contacts or unexposed controls, suggesting the specificity of the response. Distinct repertoires of serum and culture supernatant anti-M.leprae antibodies were observed when Western-blotted antigens were probed after two-dimensional gel electrophoresis. This method for assay of specific antibody production against individual components present in a complex mixture of antigens after polyclonal activation in vitro may be used to study the regulation of B cell activation in leprosy and other diseases.     

Role of neutrophil leukocytes in cutaneous infection caused by Staphylococcus aureus

Despite the high prevalence of cutaneous infections, little is known about the role of host immune responsiveness during Staphylococcus aureus dermatitis. We have recently described a murine model of infectious dermatitis induced by superantigen-producing S. aureus. To assess the role of neutrophils in staphylococcal dermatitis, mice were given granulocyte-depleting monoclonal antibody prior to and on several occasions following intracutaneous inoculation with staphylococci. The granulocyte-depleted mice that had been intradermally inoculated with S. aureus developed crusted ulcerations which tended not to heal, whereas animals injected with control monoclonal antibody displayed only minor and transient skin lesions. The finding of severe ulcerations in neutropenic mice correlated with a significantly higher burden of bacteria in the blood and skin during the early phase of the infection. Importantly, while mice with an intact granulocyte population showed only limited skin infection, bacteremia occurred in the great majority of the neutrophil-depleted animals. As a consequence, the latter individuals exhibited significantly increased levels of the proinflammatory cytokine interleukin-6 and specific antibodies to staphylococcal cell wall components and toxic shock syndrome toxin-1 in the serum. Our data point to a crucial protective role of granulocytes in S. aureus dermatitis.     

Protein A-Specific Monoclonal Antibodies and Prevention of Staphylococcus aureus Disease in Mice

Staphylococcus aureus is a leading cause of human soft tissue infections and bacterial sepsis. The emergence of antibiotic-resistant strains (methicillin-resistant S. aureus [MRSA]) has prompted research into staphylococcal vaccines and preventive measures. The envelope of S. aureus is decorated with staphylococcal protein A (SpA), which captures the Fcγ portion of immunoglobulins to prevent opsonophagocytosis and associates with the Fab portion of V(H)3-type B cell receptors to trigger B cell superantigen activity. Nontoxigenic protein A (SpA(KKAA)), when used as an immunogen in mice, stimulates humoral immune responses that neutralize the Fcγ and the V(H)3(+) Fab binding activities of SpA and provide protection from staphylococcal abscess formation in mice. Here, we isolated monoclonal antibodies (MAbs) against SpA(KKAA) that, by binding to the triple-helical bundle fold of its immunoglobulin binding domains (IgBDs), neutralize the Fcγ and Fab binding activities of SpA. SpA(KKAA) MAbs promoted opsonophagocytic killing of MRSA in mouse and human blood, provided protection from abscess formation, and stimulated pathogen-specific immune responses in a mouse model of staphylococcal disease. Thus, SpA(KKAA) MAbs may be useful for the prevention and therapy of staphylococcal disease in humans.     

Analysis of human self-reactive antibody repertoires by quantitative immunoblotting

We review the use of a quantitative immunoblotting technique to characterize human self-reactive antibody repertoires in health and disease. The interactions of plasma IgM and IgG with tissue extracts as sources of self-antigens were analyzed by quantitative immunoblotting. Data were compared by means of multiparametric statistical analysis. The data summarized here demonstrate that natural self-reactive antibody repertoires of healthy individuals are restricted to a limited subset of immunodominant autoantigens that is selected early in development, and remains conserved between individuals through ageing. The selection of human natural self-reactive IgG antibody repertoires requires normal T-/B-cell interactions. The immunoblotting assay has the potential to distinguish between autoimmune diseases with organ-related oligoclonal expansion of self-reactive clones and those characterized by broad alterations of immunoregulation. However, organ-specific autoimmune diseases may be characterized by altered patterns of antibody repertoires unrelated to the target organ. The assay also revealed an unexpected defect in the regulatory function of self-reactive IgM on the expression of self-reactive IgG repertoires in several systemic and organ-specific autoimmune diseases. The results are discussed in the light of our current understanding of the processes of selection of self-reactive B-cells and the pathophysiology of autoimmunity.     

Recent developments in SARS-CoV-2 vaccines: A systematic review of the current studies

Designing and manufacturing efficient vaccines against coronavirus disease 2019 (COVID-19) is a major objective. In this systematic review, we aimed to evaluate the most important vaccines under construction worldwide, their efficiencies and clinical results in healthy individuals and in those with specific underlying diseases. We conducted a comprehensive search in PubMed, Scopus, EMBASE, and Web of Sciences by 1 December 2021 to identify published research studies. The inclusion criteria were publications that evaluated the immune responses and safety of COVID-19 vaccines in healthy individuals and in those with pre-existing diseases. We also searched the VAERS database to estimate the incidence of adverse events of special interest (AESI) post COVID-19 vaccination. Almost all investigated vaccines were well tolerated and developed good levels of both humoural and cellular responses. A protective and efficient humoural immune response develops after the second or third dose of vaccine and a longer interval (about 28 days) between the first and second injections of vaccine could induce higher antibody responses. The vaccines were less immunogenic in immunocompromised patients, particularly those with haematological malignancies. In addition, we found that venous and arterial thrombotic events, Bell's palsy, and myocarditis/pericarditis were the most common AESI. The results showed the potency of the SARS-CoV-2 vaccines to protect subjects against disease. The provision of further effective and safe vaccines is necessary in order to reach a high coverage of immunisation programs across the globe and to provide protection against infection itself.     

Autoreactive antibody repertoire is perturbed in atherosclerotic patients

In patients with clinical symptoms of coronary atherosclerosis, T cells are activated and directed to autologous proteins contained in the active plaques, suggesting that autoimmune responses may play a role in atherosclerosis progression. Organ-specific autoimmune diseases are sometimes accompanied by broad alterations of serum autoreactive antibody repertoires. We thus investigated antibody repertoires at a global level, using a technique of immunoblotting that allows for the quantitative screening of antibody reactivities in complex antibody mixtures toward a large panel of antigens derived from homologous tissue extracts, followed by multiparametric statistical analysis of the data. We analyzed the autoreactive IgG repertoire in 20 patients with documented coronary atherosclerosis and in 20 matched healthy controls. Total proteins from atherosclerotic carotid specimens and normal arterial tissues (target organs) and from kidney, liver, and stomach (non-target control organs) were used as panels of antigens. Patients had a significantly perturbed antibody repertoire and an enhanced autoreactivity of IgG to target and non-target organs, as compared with controls. Reactivity of purified IgG to plaque and normal artery proteins was greater in patients, but reactivity of IgG in the whole serum toward normal arterial tissue was lower than in controls; this suggests that, in patients, autoreactivity toward normal arteries is regulated by serum factors. Our data indicate that atherosclerotic patients develop a perturbed humoral immune response directed toward arterial proteins, which impacts on the overall autoreactive repertoire. These findings further substantiate that autoimmune processes take place in atherosclerosis and most likely influence disease progression.     

Idiopathic Membranous Glomerulonephritis Is Associated with Altered Patterns of Self-reactive IgM and IgG Antibody Repertoires

Idiopathic membranous glomerulonephritis (MGN) is an immune complex nephropathy characterized by the subepithelial deposition of immunoglobulin (Ig)G. The pathogenesis of the disease remains largely unknown, but recent evidence suggests that human MGN may involve an autoimmune component. In the present study, we have analyzed the IgM and IgG antibody repertoires of patients with MGN towards self- and nonself-antigens using a technique of quantitative immunoblotting on a panel of whole human tissue or solubilized bacterial cell extracts as sources of antigens. Data were compared by means of multiparametric statistical analysis. We demonstrate that the antibody repertoires of self-reactive IgM and IgG in plasma of patients with MGN exhibit significantly altered patterns of reactivity, as compared with those of healthy controls. In contrast, multiparametric statistical analysis does not discriminate the reactivity patterns of IgM and IgG in plasma of patients and healthy controls towards nonself antigens. These observations indicate that a failure in the regulation of physiological self-reactivity is associated with immune complex nephropathy in MGN.     

Red blood cell transfusions are associated with alterations in self-reactive antibody repertoires of plasma IgM and IgG,independent of the presence of a specific immune response toward RBC antigens

Immunization to allogeneic RBC antigens occurs in transfused patients, and may be associated with the development of RBC-destructive antibodies directed against autologous RBC. The present study investigates the effect of transfusion of allogeneic RBC on self-reactive antibody repertoires, independent of the presence of a specific immune response directed toward RBC antigens. Antibody repertoires of IgM and IgG in plasma of hematological patients prior to starting a transfusion regime and of hematological patients receiving regular transfusions of leukocyte-depleted RBC were analyzed using quantitative immunoblotting on a panel of antigens derived from tissue and cell extracts, followed by multiparametric statistical treatment of the data. Multiparametric statistical analysis discriminated between self-reactive antibody repertoires of patients prior to starting a transfusion regime and those receiving regular RBC transfusions (IgM, 0.0001 < P < 0.0244/IgG, 0.0002 < P < 0.0088, depending on the tissue extract), whereas antibody repertoires of plasma IgM and IgG toward bacterial antigens were similar between groups of patients (P > 0.05). We conclude that the challenge of the immune system with allogeneic RBC antigens influences self-reactive antibody repertoires of plasma IgM and IgG, independent of the presence of a specific immune response toward RBC antigens.