Prevalence of antibodies against SARS-CoV-2 in the Norwegian population, August 2021.

BACKGROUND: One year into the COVID-19 pandemic, the cumulative number of confirmed COVID-19 cases in Norway was still low. In January 2021, when the Norwegian COVID-19 vaccination campaign started, the national seroprevalence estimate of SARS-CoV-2 antibodies was 3.2%. We have conducted a nationwide cross-sectional study in August 2021 to investigate the overall prevalence of SARS-CoV-2 antibodies in Norway after 8 months of COVID-19 mass vaccination and a third wave of SARS-CoV-2 infection. METHODS: Residual sera were collected from laboratories across Norway in August 2021. In IgG antibodies against the spike protein, the spike receptor binding domain (RBD) and the nucleocapsid protein of SARS-CoV-2 were measured by a bead-based flow cytometric assay. RESULTS: In total, 1926 residual sera were collected from individuals aged 0-98 years; 55.1% were from women. The overall national estimated seroprevalence from vaccination and/or infection was 62.6% (credible interval [CrI] 60.1%-65.2%) based on having antibodies against both spike and RBD. Estimated seroprevalence increased with age. Among all samples, 11.7% had antibodies against nucleocapsid. For unvaccinated children <12 years, the seroprevalence estimate due to SARS-CoV-2 infection was 12.5% (95% CrI 9.3%-16.1%). Of seropositive samples from the unvaccinated children, 31.9% lacked anti-nucleocapsid antibodies. CONCLUSIONS: The high overall SARS-CoV-2 seroprevalence estimates are in line with Norwegian registry data. Vaccination, not infection, contributed the most to the high seroprevalence in August 2021. Lack of antibodies against nucleocapsid should not automatically be interpreted as absence of previous infection as this could lead to underestimation of COVID-19 cases in seroprevalence studies.

Laboratory Methods for Detection of Infectious Agents and Serological Response in Humans With Tick-Borne Infections: A Systematic Review of Evaluations Based on Clinical Patient Samples.

Background: For the most important and well-known infections spread by Ixodes ticks, Lyme borreliosis (LB) and tick-borne encephalitis (TBE), there are recommendations for diagnosis and management available from several health authorities and professional medical networks. However, other tick-borne microorganisms with potential to cause human disease are less known and clear recommendations on diagnosis and management are scarce. Therefore, we performed a systematic review of published studies and reviews focusing on evaluation of laboratory methods for clinical diagnosis of human tick-borne diseases (TBDs), other than acute LB and TBE. The specific aim was to evaluate the scientific support for laboratory diagnosis of human granulocytic anaplasmosis, rickettsiosis, neoehrlichiosis, babesiosis, hard tick relapsing fever, tularemia and bartonellosis, as well as tick-borne co-infections and persistent LB in spite of recommended standard antibiotic treatment. Methods: We performed a systematic literature search in 11 databases for research published from 2007 through 2017, and categorized potentially relevant references according to the predefined infections and study design. An expert group assessed the relevance and eligibility and reviewed the articles according to the QUADAS (diagnostic studies) or AMSTAR (systematic reviews) protocols, respectively. Clinical evaluations of one or several diagnostic tests and systematic reviews were included. Case reports, non-human studies and articles published in other languages than English were excluded. Results: A total of 48 studies fulfilled the inclusion criteria for evaluation. The majority of these studies were based on small sample sizes. There were no eligible studies for evaluation of tick-borne co-infections or for persistent LB after antibiotic treatment. Conclusions: Our findings highlight the need for larger evaluations of laboratory tests using clinical samples from well-defined cases taken at different time-points during the course of the diseases. Since the diseases occur at a relatively low frequency, single-center cross-sectional studies are practically not feasible, but multi-center case control studies could be a way forward.

Pertussis seroprevalence among adults of reproductive age (20-39 years) in fourteen European countries.

The reported incidence of pertussis in European countries varies considerably. We aimed to study specific Bordetella pertussis seroprevalence in Europe by measuring serum IgG antibody levels to pertussis toxin (anti-PT IgG). Fourteen national laboratories participated in this study including Belgium, Denmark, Finland, Greece, Hungary, Italy, Lithuania, Malta, Norway, Poland, Portugal, Romania, Spain, and Sweden. Each country collected approximately 250 samples (N = 7903) from the age groups 20-29 years (N = 3976) and 30-39 years (N = 3927) during 2010-2013. Samples were anonymous residual sera from diagnostic laboratories and were analyzed at the national laboratories by a Swedish reference method, a commercial ELISA kit, or were sent to Sweden for analysis. The median anti-PT IgG concentrations ranged from 4 to 13.6 IU/mL. The proportion of samples with anti-PT IgG ≥100 IU/mL, indicating a recent infection ranged from 0.2% (Hungary) to 5.7% (Portugal). The highest proportion of sera with anti-PT IgG levels between 50 and <100 IU/mL, indicating an infection within the last few years, was found in Portugal (12.3%) and Italy (13.9%). This study shows that the circulation of B. pertussis is quite extensive in adults, aged 20-39 years, despite well-established vaccination programs in Europe.

Subjective health complaints and exposure to tick-borne infections in southern Norway.

OBJECTIVES: Whether tick-borne infections can cause chronic subjective health complaints is heavily debated. If such a causal connection exists, one would expect to find more health complaints among individuals exposed to tick-borne infections than among non-exposed. In this study, we aimed to assess if exposure to tick-borne infections earlier in life, evaluated by examination of serum for IgG antibodies to tick-borne microbes, was associated with self-reported somatic symptom load. MATERIALS & METHODS: All individuals with residential address in Søgne municipality in southern Norway, aged 18-69 years, were invited to participate in the study. Blood samples were analyzed for IgG antibodies to different tick-borne microbes, and somatic symptom load was charted by the Patient Health Questionnaire-15 (PHQ-15). RESULTS: Out of 7424 invited individuals, 2968 (40.0%) were included in the study. We detected IgG antibodies to Borrelia burgdorferi sensu lato (Bb) in 22.9% (95% CI 21.4-24.4). Bb seropositive individuals reported less frequently moderate to severe somatic symptom load (ie, PHQ-15 sum score ≥ 10) than seronegative individuals (12.5% versus 17.7%, difference 5.2% [95% 2.1-8.0]). However, when adjusting for several other variables in a multivariable linear regression model, presence of serum IgG antibodies to Bb was not associated with somatic symptom load. Presence of IgG antibodies to other tick-borne microbes than Bb, or seropositivity to at least two microbes, was also not associated with somatic symptom load. CONCLUSION: Presence of serum IgG antibodies to tick-borne microbes was not associated with self-reported somatic symptom load.

Human seroprevalence of antibodies to tick-borne microbes in southern Norway.

The tick Ixodes ricinus is widespread along the coastline of southern Norway, but data on human exposure to tick-borne microbes are scarce. We aimed to assess the seroprevalence of IgG antibodies to various tick-borne microbes in the general adult population living in a Norwegian municipality where ticks are abundant. Søgne is a coastline municipality in the southernmost part of Norway, and has a high density of ticks. All individuals aged 18-69 years with residential address in Søgne municipality (n = 7424) were invited to give a blood sample and answer a questionnaire. Blood samples from 3568 individuals were available for analysis. All samples were analyzed for IgG antibodies to Borrelia burgdorferi sensu lato (Bbsl), and around 1500 samples for IgG antibodies to other tick-borne microbes. Serum IgG antibodies to Bbsl were present in 22.0% (785/3568) of the tested samples, tick-borne encephalitis virus (TBEV) in 3.1% (45/1453), Anaplasma phagocytophilum in 11.0% (159/1452), Babesia microti in 2.1% (33/1537), Bartonella henselae/B. quintana in 0.1% (2/1451) and Rickettsia helvetica/R. conorii in 4.2% (60/1445). Serum IgG antibodies to A. phagocytophilum and R. helvetica/R. conorii were significantly more prevalent (p = 0.010 and p = 0.016, respectively) among individuals with serum IgG antibodies to Bbsl than among individuals without. In conclusion, our study showed a high exposure to Bbsl in the general adult population living in a coastline municipality in the southernmost part of Norway. The population is also exposed to A. phagocytophilum, R. helvetica/R. conorii, B. microti and TBEV, but very rarely B. henselae/B. quintana.

A Monoclonal Antibody against 6-Acetylmorphine Protects Female Mice Offspring from Adverse Behavioral Effects Induced by Prenatal Heroin Exposure.

Escalating opioid use among fertile women has increased the number of children being exposed to opioids during fetal life. Furthermore, accumulating evidence links prenatal opioid exposure, including opioid maintenance treatment, to long-term negative effects on cognition and behavior, and presses the need to explore novel treatment strategies for pregnant opioid users. The present study examined the potential of a monoclonal antibody (mAb) targeting heroin's first metabolite, 6-acetylmorphine (6-AM), in providing fetal protection against harmful effects of prenatal heroin exposure in mice. First, we examined anti-6-AM mAb's ability to block materno-fetal transfer of active metabolites after maternal heroin administration. Next, we studied whether maternal mAb pretreatment could prevent adverse effects in neonatal and adolescent offspring exposed to intrauterine heroin (3 × 1.05 mg/kg). Anti-6-AM mAb pretreatment of pregnant dams profoundly reduced the distribution of active heroin metabolites to the fetal brain. Furthermore, maternal mAb administration prevented hyperactivity and drug sensitization in adolescent female offspring prenatally exposed to heroin. Our findings demonstrate that passive immunization with a 6-AM-specific antibody during pregnancy provides fetal neuroprotection against heroin metabolites, and thereby prevents persistent adverse behavioral effects in the offspring. An immunotherapeutic approach to protect the fetus against long-term effects of prenatal drug exposure has not been reported previously, and should be further explored as prophylactic treatment of pregnant heroin users susceptible to relapse.

Are the current notification criteria for Lyme borreliosis in Norway suitable? Results of an evaluation of Lyme borreliosis surveillance in Norway, 1995-2013.

BACKGROUND: The approach to surveillance of Lyme borreliosis varies between countries, depending on the purpose of the surveillance system and the notification criteria used, which prevents direct comparison of national data. In Norway, Lyme borreliosis is notifiable to the Surveillance System for Communicable Diseases (MSIS). The current notification criteria include a combination of clinical and laboratory results for borrelia infection (excluding Erythema migrans) but there are indications that these criteria are not followed consistently by clinicians and by laboratories. Therefore, an evaluation of Lyme borreliosis surveillance in Norway was conducted to describe the purpose of the system and to assess the suitability of the current notification criteria in order to identify areas for improvement. METHODS: The CDC Guidelines for Evaluation of Surveillance Systems were used to develop the assessment of the data quality, representativeness and acceptability of MSIS for surveillance of Lyme borreliosis. Data quality was assessed through a review of data from 1996 to 2013 in MSIS and a linkage of MSIS data from 2008 to 2012 with data from the Norwegian Patient Registry (NPR). Representativeness and acceptability were assessed through a survey sent to 23 diagnostic laboratories. RESULTS: Completeness of key variables for cases reported to MSIS was high, except for geographical location of exposureThe NPR-MSIS linkage identified 1047 cases in both registries, while 363 were only reported to MSIS and 3914 were only recorded in NPR. A higher proportion of cases found in both registries were recorded as neuroborreliosis in MSIS (84.4 %) than those cases found only in MSIS (20.1 %). The trend (average yearly increase or decrease in reported cases) of neuroborreliosis in MSIS was not significantly different from the trend for all other clinical manifestations recorded in MSIS in negative binomial regression (p = 0.3). The 16 surveyed laboratories (response proportion 70 %) indicated differences in testing practices and low acceptability of the notification criteria. CONCLUSIONS: Given the challenges associated with diagnosing Lyme borreliosis, the selected notification criteria should be closely linked with the purpose of the surveillance system. Restricting reportable Lyme borreliosis to neuroborreliosis may increase validity, while a more sensitive case definition (potentially including erythema migrans) may better reflect the true burden of disease. We recommend revising the current notification criteria in Norway to ensure that they are unambiguous for clinicians and laboratories.

Validate or falsify: Lessons learned from a microscopy method claimed to be useful for detecting Borrelia and Babesia organisms in human blood.

BACKGROUND: A modified microscopy protocol (the LM-method) was used to demonstrate what was interpreted as Borrelia spirochetes and later also Babesia sp., in peripheral blood from patients. The method gained much publicity, but was not validated prior to publication, which became the purpose of this study using appropriate scientific methodology, including a control group. METHODS: Blood from 21 patients previously interpreted as positive for Borrelia and/or Babesia infection by the LM-method and 41 healthy controls without known history of tick bite were collected, blinded and analysed for these pathogens by microscopy in two laboratories by the LM-method and conventional method, respectively, by PCR methods in five laboratories and by serology in one laboratory. RESULTS: Microscopy by the LM-method identified structures claimed to be Borrelia- and/or Babesia in 66% of the blood samples of the patient group and in 85% in the healthy control group. Microscopy by the conventional method for Babesia only did not identify Babesia in any samples. PCR analysis detected Borrelia DNA in one sample of the patient group and in eight samples of the control group; whereas Babesia DNA was not detected in any of the blood samples using molecular methods. CONCLUSIONS: The structures interpreted as Borrelia and Babesia by the LM-method could not be verified by PCR. The method was, thus, falsified. This study underlines the importance of doing proper test validation before new or modified assays are introduced.

Geographical differences in seroprevalence of Borrelia burgdorferi antibodies in Norway, 2011-2013.

Detection of specific antibodies against Borrelia burgdorferi sensu lato is a useful aid for the diagnosis of Lyme borreliosis. However, antibodies are present in the general population. The seroprevalence increase with age, and varies according to the prevalence of infected ticks. We performed a seroprevalence study of IgM and IgG antibody reactivity against B. burgdorferi sensu lato in Norway by age-groups and geography, in order to provide a reference set of seroprevalence to inform the interpretation of positive test results. We used two commercially available enzyme immuno assays (EIA) and a multiplexed bead assay to detect Borrelia IgG antibodies in a convenience sample of 3057 sera collected from clinical chemistry laboratories in 10 of 19 counties in Norway between December 2011 and January 2013. We estimated seroprevalence by age and county by a logistic regression model. IgM antibodies were detected by two commercially available EIAs and a multiplexed bead assay. The overall seroprevalence of Borrelia IgG was 4.0% (95% CI: 2.4-6.6%) and 4.2% (2.6-6.8%) by the two EIAs, respectively. The seroprevalence increased by age, and by geography from north to south. The IgG assays showed a good agreement for positive test results. All sera positive for IgG in the multiplexed bead assay reacted with the VlsE antigen, and also had high antibody levels by EIA. The Borrelia seroprevalence varied by geography and increased by age. The results indicate regional differences in pre-test probabilities for positive test results, and can inform the interpretation of laboratory results.

Cellular Immune Responses in Humans Induced by Two Serogroup B Meningococcal Outer Membrane Vesicle Vaccines Given Separately and in Combination.

MenBvac and MeNZB are safe and efficacious outer membrane vesicle (OMV) vaccines against serogroup B meningococcal disease. Antibody responses have previously been investigated in a clinical trial with these two OMV vaccines given separately (25 µg/dose) or in combination (12.5 and 12.5 µg/dose) in three doses administered at 6-week intervals. Here, we report the results from analyzing cellular immune responses against MenBvac and MeNZB OMVs in terms of antigen-specific CD4(+)T cell proliferation and secretion of cytokines. The proliferative CD4(+)T cell responses to the combined vaccine were of the same magnitude as the homologous responses observed for each individual vaccine. The results also showed cross-reactivity in the sense that both vaccine groups receiving separate vaccines responded to both homologous and heterologous OMV antigen when assayed for antigen-specific cellular proliferation. In addition, a multiplex bead array assay was used to analyze the presence of Th1 and Th2 cytokines in cell culture supernatants. The results showed that gamma interferon, interleukin-4 (IL-4), and IL-10 responses could be detected as a result of vaccination with both the MenBvac and the MeNZB vaccines given separately, as well as when given in combination. With respect to cross-reactivity, the cytokine results paralleled the observations made for proliferation. In conclusion, the results demonstrate that cross-reactive cellular immune responses involving both Th1 and Th2 cytokines can be induced to the same extent by different tailor-made OMV vaccines given either separately or in combination with half the dose of each vaccine.

Etiology of Pneumonia in a Pediatric Population with High Pneumococcal Vaccine Coverage: A Prospective Study.

BACKGROUND: Improved Childhood Immunizations Programs, especially the introduction of pneumococcal vaccination, better diagnostic methods and the importance of reduced antibiotic misuse, make this a critical time to increase knowledge on the etiology of pediatric pneumonia. Our main objective was to identify the contribution of various microbiological species that causes pneumonia in previously healthy children and adolescents in a population with high pneumococcal conjugate vaccine coverage. METHODS: This prospective, observational study enrolled patients with clinical and radiological signs of pneumonia over a 2-year period. Both inpatients and outpatients were included. Paired sera, nasopharyngeal polymerase chain reaction and bacterial cultures from blood and pleura were analyzed to detect potential viral and bacterial causative pathogens. RESULTS: TWO HUNDRED AND SIXTY-FIVE: cases of clinical and radiological verified pneumonia were identified. The pneumococcal vaccine coverage was 85%. We identified a causative pathogen in 84.2% of all cases; 63.4% with single viral etiology, 11.3% with pneumococcus and 7.5% with mycoplasma infection. Respiratory syncytial virus was the most common pathogen in children younger than 5 years, whereas mycoplasma was the most common in older children. CONCLUSIONS: We identified the majority of 265 cases with radiology proven pneumonia as single viral infections, predominantly respiratory syncytial virus and a much lower proportion of bacterial causes. These findings may impact pneumonia management guidelines in areas where widespread pneumococcal vaccination is provided and contribute to reduced antibiotic overuse in pediatric pneumonia.

Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions.

Engineering of the constant Fc part of monoclonal human IgG1 (hIgG1) Abs is an approach to improve effector functions and clinical efficacy of next-generation IgG1-based therapeutics. A main focus in such development is tailoring of in vivo half-life and transport properties by engineering the pH-dependent interaction between IgG and the neonatal Fc receptor (FcRn), as FcRn is the main homeostatic regulator of hIgG1 half-life. However, whether such engineering affects binding to other Fc-binding molecules, such as the classical FcγRs and complement factor C1q, has not been studied in detail. These effector molecules bind to IgG1 in the lower hinge-CH2 region, structurally distant from the binding site for FcRn at the CH2-CH3 elbow region. However, alterations of the structural composition of the Fc may have long-distance effects. Indeed, in this study we show that Fc engineering of hIgG1 for altered binding to FcRn also influences binding to both the classical FcγRs and complement factor C1q, which ultimately results in alterations of cellular mechanisms such as Ab-dependent cell-mediated cytotoxicity, Ab-dependent cellular phagocytosis, and Ab-dependent complement-mediated cell lysis. Thus, engineering of the FcRn-IgG1 interaction may greatly influence effector functions, which has implications for the therapeutic efficacy and use of Fc-engineered hIgG1 variants.

Anti-pertussis antibody kinetics following DTaP-IPV booster vaccination in Norwegian children 7-8 years of age.

At the age of 7-8 years a booster of diphtheria, tetanus, acellular pertussis and polio vaccine is recommended for children in Norway. In this cross-sectional study we have analysed the antibody levels against pertussis vaccine antigens in sera from 498 children aged 6-12 years. The purposes of this study were to investigate the duration of the booster response against the pertussis vaccine antigens pertussis toxin (PT) and filamentous haemagglutinin (FHA); to determine the presence of high levels of pertussis antibodies in absence of recent vaccination; and to analyse how booster immunisation may interfere with the serological pertussis diagnostics. Prior to the booster the IgG antibody levels against PT revealed a geometric mean of 7.3IU/ml. After the booster the geometric mean peak anti-PT IgG response reached to 45.6IU/ml, followed by a steady decline in antibody levels over the next few years. The IgG anti-FHA levels followed the anti-PT IgG profiles. Three years after the booster the geometric mean IgG levels were only slightly above pre-booster levels. Prior to the booster 44% of the sera contained ≤5IU/ml of anti-PT IgG compared to18% 3 years after and 30% 4 years after the booster. When recently vaccinated children were excluded, 6.2% of the children had anti-PT IgG levels above 50IU/ml which may indicate pertussis infection within the last 2 years. This study indicates that the currently used acellular pertussis vaccines induce moderate immune responses to the pertussis antigens and that the antibodies wane within few years after the booster. This lack of sustained immune response may partly be responsible for the increased number of pertussis cases observed in this age group during the last years.

Experimental infection of healthy volunteers with enterotoxigenic Escherichia coli wild-type strain TW10598 in a hospital ward.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is an important cause of childhood diarrhea in resource-limited regions. It is also an important cause of diarrhea in travellers to these areas.To evaluate the protective efficacy of new ETEC vaccines that are under development, there is a need to increase the capacity to undertake Phase IIB (human challenge) clinical trials and to develop suitable challenge models. METHODS: An in-hospital study was performed where fasting adult volunteers were experimentally infected with 1 × 106 to 1 × 109 colony forming units (CFUs) of the wild-type ETEC strain TW10598, which had been isolated from a child with diarrhea in West Africa in 1997. We recorded symptoms and physical signs and measured serum immune response to the TW10598 bacterium. RESULTS: We included 30 volunteers with mean age 22.8 (range 19.8, 27.4) years. The most common symptoms were diarrhea (77%), abdominal pain (67%), nausea (63%), and abdominal cramping (53%). Seven subjects (23%) experienced fever, none were hypotensive. Most of the volunteers responded with a substantial rise in the level of serum IgA antibodies against the challenge strain. CONCLUSIONS: We established the capacity and methods for safely undertaking challenge studies to measure the efficacy of ETEC vaccine candidates in a hospital ward. Strain TW10598 elicited both clinical symptoms and an immune response across the doses given.

Human antibody responses to pneumococcal surface protein A and capsular polysaccharides during acute and convalescent stages of invasive disease in adult patients.

The IgG antibody responses to pneumococcal surface protein A (PspA) and capsular polysaccharides in acute and convalescent-phase sera from 10 adult patients with invasive pneumococcal disease were analysed. The relatedness between the strains were characterized by capsular serotyping (1, 4, 7F, 9V, 12F and 19F), multilocus sequence typing (MLST) and sequencing of the gene coding for PspA. Immunoblotting with the patient's own infecting strain used as whole cell antigen revealed strong antibody responses to PspA in 4 of 10 patients. Two of these patients showed cross reactivity of PspA antibodies within PspA families 1 and 2 by ELISA measurements with recombinant PspA proteins. Using ELISA, we found increased levels of capsular-specific antibodies during convalescent phase in 9 of 10 patients. All patients, except one, revealed low antibody levels in their acute phase sera. The binding of serum antibodies to live pneumococci using the patient's own infective strain was measured by flow cytometry. The antibodies binding to the live pneumococci corresponded to the serotype-specific polysaccharides by ELISA. Low antibody-binding activities to their infective strain in the acute serum may explain why they were not protected.

Immunization of teenagers with a fifth dose of reduced DTaP-IPV induces high levels of pertussis antibodies with a significant increase in opsonophagocytic activity.

Waning vaccine-induced immunity against Bordetella pertussis is observed among adolescents and adults. A high incidence of pertussis has been reported in this population, which serves as a reservoir for B. pertussis. A fifth dose of reduced antigen of diphtheria-tetanus-acellular-pertussis and inactivated polio vaccine was given as a booster dose to healthy teenagers. The antibody activity against B. pertussis antigens was measured prior to and 4 to 8 weeks after the booster by different assays: enzyme-linked immunosorbent assays (ELISAs) of IgG and IgA against pertussis toxin (PT) and filamentous hemagglutinin (FHA), IgG against pertactin (PRN), opsonophagocytic activity (OPA), and IgG binding to live B. pertussis. There was a significant increase in the IgG activity against PT, FHA, and PRN following the booster immunization (P < 0.001). The prebooster sera showed a geometric mean OPA titer of 65.1 and IgG binding to live bacteria at a geometric mean concentration of 164.9 arbitrary units (AU)/ml. Following the fifth dose, the OPA increased to a titer of 360.4, and the IgG concentration against live bacteria increased to 833.4 AU/ml (P < 0.001 for both). The correlation analyses between the different assays suggest that antibodies against FHA and PRN contribute the most to the OPA and IgG binding.

Polyreactivity of monoclonal antibodies made against human erythrocyte membranes with various pathogenic bacteria.

Glycophorins comprise the major sialoglycoproteins of the human erythrocyte membrane. Several years ago we described a murine monoclonal antibody (MAb), designated 124,D-7 (IgM), developed by in vitro immunization with human erythrocyte membranes as antigen. We found the MAb reacted with a neuraminidase-dependent epitope on glycophorin A. Recent findings using ELISA with various bacteria as coating antigens have demonstrated strong cross-reactions of MAb 124,D-7 with some bacteria like Legionella and no reaction with bacteria such as Streptococcus pneumoniae. A second MAb, 130,E-4 (IgM), generated by the in vitro immunization technique, agglutinated human red cells irrespective of blood groups. This MAb showed strong cross-reactions with bacteria different from those being positive with MAb 124,D-7. The broad cross-reactivities of the two MAbs suggested that they are polyreactive antibodies. Sequencing of the V(H) and V(L) genes of MAb 124,D-7 showed germ-like sequences characteristic of polyreactive antibodies. The nucleotide sequences of the V(H) and V(L) genes of MAb 124,D-7 matched sequences coding for antibodies against CD34 and cross-reacting streptococcal antibodies. For Legionella pneumophila, the main interacting band on immunoblots was identified as the major outer membrane protein by mass spectrometry after separation by isoelectric focusing followed by SDS-PAGE. Flow cytometry showed that the epitope for MAb 124,D-7 was not displayed on live L. pneumophila but became exposed after heat treatment. Studies with one of the control MAbs, 145,F-2, directed against phosphorylcholine, which is known to be present on erythrocytes and some bacteria, showed that the epitope is deeply buried in the human erythrocyte membrane as neither neuraminidase nor papain exposed the epitope. The positive control MAb 3/1 directed against an epitope on LPS of L. pneumophila revealed weak cross-reactions with Pseudomonas aeruginosa.

Critical roles of complement and antibodies in host defense mechanisms against Neisseria meningitidis as revealed by human complement genetic deficiencies.

Certain complement defects are associated with an increased propensity to contract Neisseria meningitidis infections. We performed detailed analyses of complement-mediated defense mechanisms against N. meningitidis 44/76 with whole blood and serum from two adult patients who were completely C2 or C5 deficient. The C5-deficient patient and the matched control were also deficient in mannose-binding lectin (MBL). The proliferation of meningococci incubated in freshly drawn whole blood was estimated by CFU and quantitative DNA real-time PCR. The serum bactericidal activity and opsonophagocytic activity by granulocytes were investigated, including heat-inactivated postvaccination sera, to examine the influence of antimeningococcal antibodies. The meningococci proliferated equally in C2- and C5-deficient blood, with a 2 log(10) increase of CFU and 4- to 5-log(10) increase in DNA copies. Proliferation was modestly decreased in reconstituted C2-deficient and control blood. After reconstitution of C5-deficient blood, all meningococci were killed, which is consistent with high antibody titers being present. The opsonophagocytic activity was strictly C2 dependent, appeared with normal serum, and increased with postvaccination serum. Serum bactericidal activity was strictly dependent on C2, C5, and high antibody titers. MBL did not influence any of the parameters observed. Complement-mediated defense against meningococci was thus dependent on the classical pathway. Some opsonophagocytic activity occurred despite low levels of antimeningococcal antibodies but was more efficient with immune sera. Serum bactericidal activity was dependent on C2, C5, and immune sera. MBL did not influence any of the parameters observed.