Complement activation is required for induction of a protective antibody response against West Nile virus infection

Infection with West Nile virus (WNV) causes a severe infection of the central nervous system (CNS) with higher levels of morbidity and mortality in the elderly and the immunocompromised. Experiments with mice have begun to define how the innate and adaptive immune responses function to limit infection. Here, we demonstrate that the complement system, a major component of innate immunity, controls WNV infection in vitro primarily in an antibody-dependent manner by neutralizing virus particles in solution and lysing WNV-infected cells. More decisively, mice that genetically lack the third component of complement or complement receptor 1 (CR1) and CR2 developed increased CNS virus burdens and were vulnerable to lethal infection at a low dose of WNV. Both C3-deficient and CR1- and CR2-deficient mice also had significant deficits in their humoral responses after infection with markedly reduced levels of specific anti-WNV immunoglobulin M (IgM) and IgG. Overall, these results suggest that complement controls WNV infection, in part through its ability to induce a protective antibody response.     

Second International Diagnostic Accuracy Study for the Serological Detection of West Nile Virus Infection

Background

In recent decades, sporadic cases and outbreaks in humans of West Nile virus (WNV) infection have increased. Serological diagnosis of WNV infection can be performed by enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA) neutralization test (NT) and by hemagglutination-inhibition assay. The aim of this study is to collect updated information regarding the performance accuracy of WNV serological diagnostics.

Methodology/Principal findings

In 2011, the European Network for the Diagnostics of Imported Viral Diseases-Collaborative Laboratory Response Network (ENIVD-CLRN) organized the second external quality assurance (EQA) study for the serological diagnosis of WNV infection. A serum panel of 13 samples (included sera reactive against WNV, plus specificity and negative controls) was sent to 48 laboratories involved in WNV diagnostics. Forty-seven of 48 laboratories from 30 countries participated in the study. Eight laboratories achieved 100% of concurrent and correct results. The main obstacle in other laboratories to achieving similar performances was the cross-reactivity of antibodies amongst heterologous flaviviruses. No differences were observed in performances of in-house and commercial test used by the laboratories. IFA was significantly more specific compared to ELISA in detecting IgG antibodies. The overall analytical sensitivity and specificity of diagnostic tests for IgM detection were 50% and 95%, respectively. In comparison, the overall sensitivity and specificity of diagnostic tests for IgG detection were 86% and 69%, respectively.

Conclusions/Significance

This EQA study demonstrates that there is still need to improve serological tests for WNV diagnosis. The low sensitivity of IgM detection suggests that there is a risk of overlooking WNV acute infections, whereas the low specificity for IgG detection demonstrates a high level of cross-reactivity with heterologous flaviviruses.     

Induction of epitope-specific neutralizing antibodies against West Nile virus

Previous studies have established that an epitope on the lateral ridge of domain III (DIII-lr) of West Nile virus (WNV) envelope (E) protein is recognized by strongly neutralizing type-specific antibodies. In contrast, an epitope against the fusion loop in domain II (DII-fl) is recognized by flavivirus cross-reactive antibodies with less neutralizing potential. Using gain- and loss-of-function E proteins and wild-type and variant WNV reporter virus particles, we evaluated the expression pattern and activity of antibodies against the DIII-lr and DII-fl epitopes in mouse and human serum after WNV infection. In mice, immunoglobulin M (IgM) antibodies to the DIII-lr epitope were detected at low levels at day 6 after infection. However, compared to IgG responses against other epitopes in DI and DII, which were readily detected at day 8, the development of IgG against DIII-lr epitope was delayed and did not appear consistently until day 15. This late time point is notable since almost all death after WNV infection in mice occurs by day 12. Nonetheless, at later time points, DIII-lr antibodies accumulated and comprised a significant fraction of the DIII-specific IgG response. In sera from infected humans, DIII-lr antibodies were detected at low levels and did not correlate with clinical outcome. In contrast, antibodies to the DII-fl were detected in all human serum samples and encompassed a significant percentage of the anti-E protein response. Our experiments suggest that the highly neutralizing DIII-lr IgG antibodies have little significant role in primary infection and that the antibody response of humans may be skewed toward the induction of cross-reactive, less-neutralizing antibodies.     

侵袭性真菌深部感染的早期实验室诊断

目的探讨假丝酵母菌甘露聚糖抗原和假丝酵母菌IgG/IgM抗体、曲霉半乳甘露聚糖抗原和烟曲霉IgG抗体在侵袭性真菌病早期临床诊断中的应用价值。方法收集已确诊侵袭性假丝酵母菌病患者18例,侵袭性烟曲霉病患者6例,单纯细菌感染患者20例,浅部真菌感染患者20例,健康体检者(正常对照组)20例,通过酶联免疫吸附法(ELISA)检测患者血清甘露聚糖和假丝酵母菌IgG/IgM抗体以及曲霉半乳甘露聚糖抗原和烟曲霉IgG抗体浓度,计算各指标的灵敏度、特异度、阳性预测值、阴性预测值和受试者工作特征(ROC)曲线下面积。结果甘露聚糖抗原和假丝酵母菌IgG/IgM抗体联合测定的敏感度为66.7%,特异度为83.3%,阴性预测值为100.0%,阳性预测值为85.7%,ROC曲线下面积为0.992(95%CI:0.974~1.000);半乳甘露聚糖抗原和烟曲霉IgG抗体联合测定的敏感度为66.7%,特异度为95.0%,阴性预测值为98.2%,阳性预测值为100.0%,ROC曲线下面积为0.978(95%CI:0.934~1.000)。结论甘露聚糖抗原和假丝酵母菌IgG/IgM抗体、半乳甘露聚糖抗原和烟曲霉IgG抗体联合检测对深部真菌感染的早期诊断具有重要意义。     

Immunodiagnosis of Dengue Virus Infection Using Saliva

Keeping in view the complications and the case fatality associated with dengue virus, several serologic tests have been developed. However, the major drawback of these serologic tests is the need for a venous blood sample obtained by invasive venipuncture. As a noninvasive alternative, saliva provides a body fluid that contains antibodies of diagnostic importance. Hence, the detection of DEN-specific IgM and IgG antibodies in serum and saliva from 80 patients was compared. Salivary IgM antibodies were detected in 100% of the serum IgM-positive samples and in 30% of the serum samples that were negative for IgM antibodies. Salivary IgG antibodies were detected in 93.3% of the serum samples that were positive for anti-dengue IgG antibodies and in none of the serum IgG-negative cases. None of the specimens from the healthy controls showed the presence of IgM or IgG antibodies. The detection of both IgG and IgM antibodies in saliva correlated well with the serum IgG and IgM detection by the ELISA test (r = 0.6322 and r = 0.4227). Detection of salivary IgM antibodies by ELISA showed 100% sensitivity, 70% specificity, 90.9% positive predictive value, and 100% negative predictive value. The detection of IgG in saliva proved to be a promising tool as the sensitivity, specificity, positive predictive value, and negative predictive value were found out to be 93.3%, 100%, 100%, and 83.3%, respectively. Thus, from this study we conclude that the detection of DEN-specific salivary IgG and IgM antibodies are useful markers for dengue infection.     

Comparison of diagnostic performances of ten different immunoassays detecting anti-CCHFV IgM and IgG antibodies from acute to subsided phases of Crimean-Congo hemorrhagic fever

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a geographically widespread tick-borne arbovirus that has been recognized by the WHO as an emerging pathogen needing urgent attention to ensure preparedness for potential outbreaks. Therefore, availability of accurate diagnostic tools for identification of acute cases is necessary.A panel comprising 121 sequential serum samples collected during acute, convalescent and subsided phase of PCR-proven CCHFV infection from 16 Kosovar patients was used to assess sensitivity. Serum samples from 60 healthy Kosovar blood donors were used to assess specificity. All samples were tested with two IgM/IgG immunofluorescence assays (IFA) from BNITM, the CCHFV Mosaic 2 IgG and IgM indirect immunofluorescence tests (IIFT) from EUROIMMUN, two BlackBox ELISAs for the detection of CCHFV-specific IgM and IgG antibodies (BNITM), two Anti-CCHFV ELISAs IgM and IgG from EUROIMMUN using recombinant structural proteins of CCHFV antigens, and two ELISAs from Vector-Best (IgM: μ-capture ELISA, IgG: indirect ELISA using immobilized CCHFV antigen). Diagnostic performances were compared between methods using sensitivity, specificity, concordance and degree of agreement with particular focus on the phase of the infection.In early and convalescent phases of infection, the sensitivities for detecting specific IgG antibodies differed for the ELISA test. The BlackBox IgG ELISA yielded the highest, followed by the EUROIMMUN IgG ELISA and finally the VectorBest IgG ELISA with the lowest sensitivities. In the subsided phase, the VectorBest IgM ELISA detected a high rate of samples that were positive for anti-CCHFV IgM antibodies. Both test systems based on immunofluorescence showed an identical sensitivity for detection of anti-CCHFV IgM antibodies in acute and convalescent phases of infection.Available serological test systems detect anti-CCHFV IgM and IgG antibodies accurately, but their diagnostic performances vary with respect to the phase of the infection.     

Printed glycan array: antibodies as probed in undiluted serum and effects of dilution

Using printed glycan array (PGA) we compared the results of antibody profiling in undiluted, moderately (1:15) and highly (1:100) diluted human blood serum. Undiluted serum is suitable for studying blood as a tissue in its native state, whereas to study the serum of newborns or small animals one usually has to dilute the starting material in order to have sufficient volume for PGA experimentation. The PGA used in this study allows for the use of whole serum without modifications to the protocol, and the background is surprisingly low. Antibodies profiles observed in undiluted serum versus 1:15 dilution were similar, with only a limited number of new signals identified in the undiluted serum. However, unexpected irregularities were found when IgG and IgM are measured separately, namely, at a 1:15 dilution more intensive IgG signals for many glycans are observed. We believe that in conditions of moderate dilution IgG and IgM antibodies can compete with each other for antigen and as a result, the higher affinity anti-glycan IgGs give rise to more intense signals. Therefore depending on the purpose, different dilutions of serum will be optimal: in competitive 1:15 conditions the observed IgG/IgM ratio corresponds to their titer, whereas at 1:100 dilution the measured ratio corresponds to real molar concentration of IgG and IgM.     

Comparison of commercially available and novel West Nile virus immunoassays for detection of seroconversion in pig-tailed macaques (Macaca nemestrina)

We report the assessment and validation of an NS1 epitope-blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to West Nile virus (WNV) in macaques. Sera from naturally infected Macaca nemestrina were tested by ELISA and plaque reduction neutralization test (PRNT). Results were correlated with hemagglutination inhibition (HAI) data. Our results demonstrate that the blocking ELISA rapidly and specifically detects WNV infection in M. nemestrina. In addition, the diagnostic value of 7 commercially available immunoassays (PanBio immunoglobulin [Ig] M ELISA, PanBio IgG ELISA, PanBio immunofluorescence assay (IFA), InBios IgG ELISA, InBios IgM ELISA, Focus Diagnostics IgG ELISA, and Focus Diagnostics IgM ELISA) in M. nemestrina was evaluated and compared with that of the epitope-blocking ELISA. The PanBio IgG ELISA was found to effectively diagnose WNV exposure in M. nemestrina. Further, PanBio IFA slides are fast and reliable screening tools for diagnosing flaviviral exposure in M. nemestrina.     

Early adaptive humoral immune responses and virus clearance in humans recently infected with pandemic 2009 H1N1 influenza virus

Few studies on the humoral immune responses in human during natural influenza infection have been reported. Here, we used serum samples from pandemic 2009 H1N1 influenza infected patients to characterize the humoral immune responses to influenza during natural infection in humans. We observed for the first time that the pandemic 2009 H1N1 influenza induced influenza A-specific IgM within days after symptoms onset, whereas the unit of IgG did not changed. The magnitude of influenza A-specific IgM antibodies might have a value in predicting the rate of virus clearance to some degree. However, the newly developed IgM was not associated with hemagglutination inhibition (HI) activities in the same samples but correlated with HI activities of subsequently collected sera which were mediated by IgG antibodies, indicating that IgM was critical for influenza infection and influences subsequent IgG antibody responses. These findings provide new important insights on the human immunity to natural influenza infection.     

CD4+ T-cell responses are required for clearance of West Nile virus from the central nervous system

Although studies have established that innate and adaptive immune responses are important in controlling West Nile virus (WNV) infection, the function of CD4(+) T lymphocytes in modulating viral pathogenesis is less well characterized. Using a mouse model, we examined the role of CD4(+) T cells in coordinating protection against WNV infection. A genetic or acquired deficiency of CD4(+) T cells resulted in a protracted WNV infection in the central nervous system (CNS) that culminated in uniform lethality by 50 days after infection. Mice surviving past day 10 had high-level persistent WNV infection in the CNS compared to wild-type mice, even 45 days following infection. The absence of CD4(+) T-cell help did not affect the kinetics of WNV infection in the spleen and serum, suggesting a role for CD4-independent clearance mechanisms in peripheral tissues. WNV-specific immunoglobulin M (IgM) levels were similar to those of wild-type mice in CD4-deficient mice early during infection but dropped approximately 20-fold at day 15 postinfection, whereas IgG levels in CD4-deficient mice were approximately 100- to 1,000-fold lower than in wild-type mice throughout the course of infection. WNV-specific CD8(+) T-cell activation and trafficking to the CNS were unaffected by the absence of CD4(+) T cells at day 9 postinfection but were markedly compromised at day 15. Our experiments suggest that the dominant protective role of CD4(+) T cells during primary WNV infection is to provide help for antibody responses and sustain WNV-specific CD8(+) T-cell responses in the CNS that enable viral clearance.     

Comparison of two microimmunofluorescence tests for detecting antibodies to Chlamydia pneumoniae

Two microimmunofluorescence (MIF) tests were compared for detection of antibodies to Chlamydia pneumoniae: the microimmunofluorescence of Washington University and the microimmunofluorescence of Chlamydia Serofia. Concordant positive results at the same dilution were observed for IgG in 37.33% of sera tested and concordant negative results were found in 44%. Variations of one fold dilution were observed in 36 sera. Extensive variations (2-3 two-fold dilutions) in the numeric titer values were observed in 20 serum samples with titers of antibody generally higher in the Chlamydia Serofia MIF than in the Washington MIF, resulting in a diagnosis of current infection in three patients. IgM were found with both methods only in one patient. The discrepancies observed may be due to several factors including the different TWAR strain used as antigen in the two tests and the dilution of the FITC-labelled conjugated anti-human IgG.We think that MIF serology may also be influenced by the type of response of the host that may depend on the "local strain" of C. pneumoniae that may express different antigens or in different amounts in comparison with the strains used by the commercial kit.     

Evaluation of the usefulness of commercial enzyme-linked immunosorbent assays for diagnosis of Mycoplasma pneumoniae infections

The usefulness of the ELISA distributed by BioChem ImmunoSystems, Medial Polska, Biomedica/Virotech and prepared in our laboratory (ELISA FH-K) for diagnosis of the M. pneumoniae infections was estimated. Eighty six serum samples obtained from 86 patients with respiratory tract infections were simultaneously tested by ELISA-IgM/-IgG and by complement fixation test which was accepted as a reference test. The highest sensitivity in relation to the CFT was displayed by the ELISA BioChem ImmunoSystems and Medial Polska (100%), slightly lower sensitivity by the ELISA Biomedica/Virotech--96.5% and ELISA FH-K--90.9% when determining mycoplasmal antibodies of IgM. The lowest sensitivity was displayed by the ELISA Biomedica/Virotech when determining antibodies of the IgG class (54.9%). The specificity of ELISA in relation to the CFT was generally higher when detecting mycoplasmal antibodies of the IgM class then of IgG class. The study demonstrated that all 4 ELISA may be used in routine serodiagnosis of M. pneumoniae infection. For the improve of sensitivity of ELISA it's recommended to measure simultaneously the level of mycoplasmal antibodies of IgM and IgG.     

Impaired virus clearance, compromised immune response and increased mortality in type 2 diabetic mice infected with west nile virus

Clinicoepidemiological data suggest that type 2 diabetes is associated with increased risk of West Nile virus encephalitis (WNVE). However, no experimental studies have elucidated the role of diabetes in WNV neuropathogenesis. Herein, we employed the db/db mouse model to understand WNV immunopathogenesis in diabetics. Nine-week old C57BL/6 WT and db/db mice were inoculated with WNV and mortality, virus burden in the periphery and brain, and antiviral defense responses were analyzed. db/db mice were highly susceptible to WNV disease, exhibited increased tissue tropism and mortality than the wild-type mice, and were unable to clear the infection. Increased and sustained WNV replication was observed in the serum, peripheral tissues and brain of db/db mice, and heightened virus replication in the periphery was correlated with enhanced neuroinvasion and replication of WNV in the brain. WNV infection in db/db mice was associated with enhanced inflammatory response and compromised antiviral immune response characterized by delayed induction of IFN-α, and significantly reduced concentrations of WNV-specific IgM and IgG antibodies. The compromised immune response in db/db mice correlated with increased viremia. These data suggest that delayed immune response coupled with failure to clear the virus leads to increased mortality in db/db mice. In conclusion, this study provides unique mechanistic insight into the immunopathogenesis of WNVE observed in diabetics and can be used to develop therapeutics for the management of WNVE among diabetic patients.     

Comparison of enzyme-linked immunosorbent and virus neutralization assays for the serology of reovirus infections in laboratory animals

An enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of reovirus-specific IgM and IgG serum antibodies in rodents, detecting reovirus group reactive antibodies, was compared with reovirus types 1, 2 or 3 specific virus neutralization (VN) assays. To this end, serum samples were collected from specific pathogen-free (SPF) BALB/c RIVM mice, after experimental infection with any of the three mammalian reovirus serotypes. The majority (seven out of twelve) of the reovirus type 1-infected and one third (two out of six) of the reovirus type 3-infected mice died within 15 days after infection, whereas all (six out of six) of the reovirus type 2-infected animals survived. Using reovirus type 2 antigen in the ELISA, reovirus-specific IgM antibodies could be demonstrated within 1 week after infection in all the mice infected with reovirus types 2 or 3 and in the majority of the mice infected with type 1. Similarly, reovirus-specific IgG could be detected within 3 weeks in all the surviving mice infected with reovirus types 2 or 3 and within 5 weeks in all surviving mice infected with reovirus type 1. These results coincided well with the data obtained in the VN assays, in which all the infected animals also could be detected within 1 week after infection. As expected, titers were detected most rapidly and to the highest levels in the homologous VN assays. Given the sensitivity and the specificity of the ELISA system demonstrated in this paper and its suitability for incorporation in automated systems, the ELISA system should be considered valuable for the routine serologic diagnosis of reovirus infections in rodents.     

NS1 protein secretion during the acute phase of West Nile virus infection

The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNV NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.     

Time gap between oocyst shedding and antibody responses in mice infected with Cryptosporidium parvum

We observed the time gap between oocyst shedding and antibody responses in mice (3-week-old C57BL/6J females) infected with Cryptosporidium parvum. Oocyst shedding was verified by modified acid-fast staining. The individually collected mouse sera were assessed for C. parvum IgM and IgG antibodies by enzyme-linked immunosorbent assay from 5 to 25 weeks after infection. The results showed that C. parvum oocysts were shed from day 5 to 51 post-infection (PI). The IgM antibody titers to C. parvum peaked at week 5 PI, whereas the IgG antibody titers achieved maximum levels at week 25 PI. The results revealed that IgM responses to C. parvum infection occurred during the early stage of infection and overlapped with the oocyst shedding period, whereas IgG responses occurred during the late stage and was not correlated with oocyst shedding. Hence, IgM antibody detection may prove helpful for the diagnosis of acute cryptosporidiosis, and IgG antibody detection may prove effective for the detection of past infection and endemicity.     

CD40-CD40 ligand interactions promote trafficking of CD8+ T cells into the brain and protection against West Nile virus encephalitis

Recent studies have established a protective role for T cells during primary West Nile virus (WNV) infection. Binding of CD40 by CD40 ligand (CD40L) on activated CD4+ T cells provides an important costimulatory signal for immunoglobulin class switching, antibody affinity maturation, and priming of CD8+ T-cell responses. We examined here the function of CD40-dependent interactions in limiting primary WNV infection. Compared to congenic wild-type mice, CD40(-/-) mice uniformly succumbed to WNV infection. Although CD40(-/-) mice produced low levels of WNV-specific immunoglobulin M (IgM) and IgG, viral clearance from the spleen and serum was not altered, and CD8+ T-cell priming in peripheral lymphoid tissues was normal. Unexpectedly, CD8+ T-cell trafficking to the central nervous system (CNS) was markedly impaired in CD40(-/-) mice, and this correlated with elevated WNV titers in the CNS and death. In the brains of CD40(-/-) mice, T cells were retained in the perivascular space and did not migrate into the parenchyma, the predominant site of WNV infection. In contrast, in wild-type mice, T cells trafficked to the site of infection in neurons. Beside its role in maturation of antibody responses, our experiments suggest a novel function of CD40-CD40L interactions: to facilitate T-cell migration across the blood-brain barrier to control WNV infection.     

The presence of antibody in mice chronically infected with Schistosoma mansoni which blocks in vitro killing of schistosomula

We have previously reported that IgM monoclonal antibodies (mAb) that recognize surface carbohydrate determinants shared between schistosomula, cercariae, and miracidia block antibody/complement dependent killing of schistosomula in vitro. Binding assays that make use of one of the IgM mAb labeled with 125I demonstrated that serum from chronically infected mice (CMS) contained high levels of competing antibody, whereas serum from mice vaccinated with irradiated cercariae (VMS) contained little antibody of this specificity. Absorption of CMS with cercariae that removed antibodies to schistosomulum surface carbohydrate determinants increased its ability to kill schistosomula in vitro; absorption of VMS with cercariae failed to alter the lethal activity of the serum. Furthermore, fractionation of CMS by protein A Sepharose chromatography demonstrated that the IgG fraction had an increased lethal activity compared with unfractionated serum; this result was not seen with VMS. Finally, the IgM fraction of CMS was shown to block in vitro killing of the IgG fractions of both CMS and VMS. These data suggest that the blocking activities observed with the IgM mAb are contained within the serum of chronically infected mice but not in the serum of mice vaccinated with irradiated cercariae.