Detection Of Antiviral Antibodies Research Articles

MO1016: Features of the Formation of Post-Infectious and Post-Vaccinal Humoral Immune Response to SARS-COV-2 in Kidney Recipients

BACKGROUNDThis paper presents the results of studying the characteristics of the antibody response in kidney recipients who are at high risk of severe COVID-19.METHODThe study of features of the formation of post-infectious anti-SARS-CoV-2 IgG was carried out in the group of kidney recipients (n = 171) with PCR confirmed diagnosis of COVID-19. Studies of the characteristics of post-vaccination immunity were carried out in a group of vaccinated recipients (n = 49) with Sputnik V (Russia) or Vero Cell (China).ELISA was used to detect IgG to S and N proteins of the SARS-CoV-2.Comparative studies used a simple randomized selection of immunocompetent COVID-19 patients (n = 163).Statistical data processing was carried out using the χ2 and Wald's methods.RESULTSIt was found that 30 days after the onset of clinical symptoms of COVID-19 in kidney recipients, IgG to S and/or N proteins of SARS-CoV-2 were detected in 89.5% (83.9%–93.3%) of them. The detection rate of IgG to the S protein was higher than that to the N protein [87.7% (81.9%–91.9%) and 62.0% (54.5%–68.9%), respectively). At the same time, the seroprevalence to the pathogen varied by age: in the 18–34-year-old group it was 77.7% (59.9%–88.9%), in the 50–64-year-old group it was 96.7% (88.2%–99.8%) and in the group >64 years old it was 80.0% (66.8%–89.0%). This trend of antibody production in older recipients correlated with the highest frequency of registration in them of moderate and severe forms [66.7% (56.4%–75.6%)]. Differences due to the severity of the disease were noted both in the frequency of detectable antibodies [80.3% (69.5%–88.0%) in recipients with a mild form of COVID-19 and 96.0% in recipients with a severe form of infection (P < .001)] and in the intensity of the formed anti-SARS-CoV-2 immunity. Thus, high values of PC (positivity coefficient) (>12) to S protein were recorded in 52.7% (39.8%–65.3%) and 63.2% (53.1%–72.2%) patients with mild and severe forms of COVID-19, respectively, which indicated a direct dependence of the production of antiviral antibodies on the severity of the infection. It was found that in 62.6% (55.1%–69.5%) of recovered recipients anti-SARS-CoV-2 IgG persisted for a period of 3 months from the onset of infection. In a significant proportion of recipients [42.8% (35.5%–50.2%)], antibodies were detected for up to 15 months. In general, the post-infectious antibody response in kidney recipients and immunocompetent patients had similar patterns of development. Despite the general mechanism of antibody production, in immunocompetent patients, the frequency of detection of antiviral antibodies (to N protein: 82.9% and to S protein: 91.2%), tension indicators (high values of PC to N protein: 51.0% and to S protein: 75.5%) and the duration of retention (in 50.3% at 15 months of monitoring) were slightly higher than the same parameters in recipients with COVID-19.In kidney recipients after immunization with Sputnik V and Vero Cell (n = 34), a rather low detection rate of antiviral IgG [52.9% (36.7%–68.6%) compared with a similar parameter (P < .001) in vaccinated immunocompetent individuals [96.8% (94.8%–98.1%)] was found. The seroprevalence in the group of recipients with hybrid immunity (after illness and vaccination, n = 15) was 86.7% (60.9%–97.5%). In a comparative analysis of the intensity of post-vaccination immunity, high values of PC to S protein (>12) were recorded in 44.0% (26.7%–62.9%) of recipients vaccinated with Sputnik V and 50.0% (31.4%–68.6%) of recipients vaccinated with Vero Cell. The inverse relationship was observed in immunocompetent individuals: 64.7% (58.1%–70.7%) for Sputnik V and 44.2% (36.8%–51.8%] for Vero Cell.CONCLUSIONThe patterns of antibody response to the causative agent in recipients with COVID-19 are comparable to those in immunocompetent patients, while for vaccinated recipients, a low frequency of detection of antiviral antibodies was shown, which indicates the need to continue research on the humoral immunity in people with vulnerable immunity in order to select the best tactics for COVID-19 immunization.

Sensitive detection systems for infectious agents in xenotransplantation.

Xenotransplantation of pig cells, tissues, or organs may be associated with transmission of porcine microorganisms, first of all of viruses, to the transplant recipient, potentially inducing a disease (zoonosis). I would like to define detection systems as the complex of sample generation, sample preparation, sample origin, time of sampling, and the necessary negative and positive controls along with the specific detection methods, either PCR-based, cell-based, or immunological methods. Some xenotransplantation-relevant viruses have already been defined; others are still unknown. The PCR-based methods include PCR and real-time PCR for DNA viruses, and RT-PCR and real-time RT-PCR for RNA viruses as well as for virus expression studies at the RNA level. Furthermore, droplet digital PCR (ddPCR) can be used for the determination of virus and provirus copies. To detect expression at the protein level, immunofluorescence, immunohistochemistry, and Western blot analyses can be used. To detect virus production and to detect infectious viruses, electron microscopy and infection assays can be used. Furthermore, immunological methods such as Western blot analysis or ELISA can be used to detect virus-specific antibodies. Detection of antiviral antibodies is a reliable and sensitive indirect detection method. For these immunological methods, purified viruses, recombinant viral proteins, or synthetic peptides are used as antigens and control sera and control antigens are needed. All these methods have been used in the past for the characterization of different pig breeds including genetically modified pigs generated for xenotransplantation and for the screening of recipients in preclinical and clinical xenotransplantations. Whereas in preclinical trials a few porcine viruses have been transmitted to the non-human primate recipients, in first clinical trials no such transmissions to humans were observed. Further improvement of the detection systems and their application in virus elimination programs will lead to clean donor animals and a safe xenotransplantation.

Detection of anti-viral antibodies from meat juice of wild boars

Wild boars are a reservoir for many zoonotic pathogens and a good sentinel for surveillance of zoonotic viral infections, but collection of serum samples from wild boars in the field is sometimes difficult and requires special equipment and techniques. In this study, ELISA using meat juices extracted from the heart and diaphragm of wild boars, instead of serum samples, was performed to detect antibodies against zoonotic pathogens, Japanese encephalitis virus and hepatitis E virus. The results of ELISA using meat juice samples were significantly correlated with those using serum samples and meat juice contained one-fifth the antibodies of serum samples. As meat juice is easily collected from wild animals in the field without special equipment and techniques, ELISA using meat juice is a simple and superior method for serological survey of zoonosis among wild animals.

Evaluation of testing strategies for reliable measurement of rates of subclinical mosquito-borne viral infections

Aim Insect-transmitted viral infections remain a significant public health concern in Australia. The positive predictive value (PPV) for detection of anti-viral antibodies in donors was assessed using commercially-available kits. Methods Blood samples collected from Australian blood donors were tested by ELISA for IgM or IgG specific for either Dengue virus (DENV), Ross River virus (RRV) or Barmah Forest virus (BFV). The PPV was determined by comparison with results from reference laboratories. Results For the commercially-available kits: anti-DENV IgG, 225 kit-positive, 213 confirmed, PPV 94.7%; anti-RRV IgG, 315 kit-positive, 239 confirmed, PPV 75.9%; anti-BFV IgG, 127 kit-positive, 47 confirmed, PPV 37.0%; anti-DENV IgM, 118 kit-positive, 7 confirmed, PPV 5.9%; anti-RRV IgM, 138 kit-positive, 45 confirmed, PPV 32.6%; anti-BFV IgM, 142 kit-positive, 71 confirmed, PPV 50.0%. Some potential sources of unexpected kit-positives were investigated, however, these contributed minimally to the unacceptable PPVs. Conclusions These kits are designed for diagnostic testing of symptomatic patients, however, it would be expected that reliability for investigation of sub-clinical infection would be equal. PPVs, with the exception of 94.7% for IgG anti-DENV, were less than 80%. To measure rates of subclinical infection an investigation algorithm including exploration of unexpected positives and reliable confirmatory testing is required.

From XMRV to HIV-1 and back

Xenotropic MLV-related retrovirus (XMRV) was first reported about 5 years ago in a few cases of prostate cancer, but did not attract much attention until its reported association with a large fraction of chronic fatigue syndrome cases about 2 years ago. The publication of the XMRV-CFS connection created a ripple of excitement and interest in the scientific, medical, and patient communities reminiscent of the reports of the association of another retrovirus—HIV-with AIDS some 25 years previously. Although I was not directly involved in either discovery at the time, I was a very interested observer who had been working with various retroviruses and who subsequently became involved in both HIV and research, and I will offer some observations on the parallels and differences between the two. It is noteworthy, that, within 2 years of the first report, in 1983, of the virus we now call HIV, the key findings were widely replicated by virologists using a variety of different assays. By contrast, most of the results of the paper - isolation of infectious virus from patients, frequent detection of virus in plasma and PBMCs by PCR, detection of antiviral antibodies - remain to be replicated outside of the laboratories that authored the original report despite considerable effort worldwide. HIV is now known as the cause of a global pandemic responsible for large numbers of deaths annually and considerable hardship and economic loss. By contrast, is considered by most virologists to be the consequence of a collection of artifacts originating from endogenous murine leukemia viruses prevalent in laboratory and wild mice. In this talk, I will discuss our recent work that uncovered how these associations arose and how even experienced retrovirologists were misled by the initial reports. There are several related, but distinct, issues that need to be considered. First, various mouse (Mus musculus) subspecies carry over a hundred different endogenous proviruses closely (>90%) related to in their DNA. Second, mice are extremely widespread, as is their DNA, which can be found sporadically on laboratory surfaces, as well as contaminants of common reagents and materials. Sensitive PCR assays can detect XMRV related sequences in DNA from tiny fractions of one cell. To detect such contamination, we developed a more sensitive assay based on mouse IAP sequences present in thousands of copies per cell. Third, although only a few of the endogenous MLV proviruses encode infectious virus, it has been known since the 1970s that some of them can give rise to virus that can infect human tumor lines when passaged through nude mice. Indeed, A virus identical to is produced by the 22RV1 prostate cancer line that was derived in just this way In initial reports, however, did not appear to be sufficiently similar to known proviruses to have been derived this way. However, we have recently shown that this is exactly how it did arise, but not from infection of the precursor CWR22 xenograft with a single virus, but rather with a recombinant between the progeny of two previously undescribed proviruses found in the nude mice used for passage. Since the predicted recombinant is ancestral to all isolates, and cannot have arisen more than once, it must have found its way into many laboratories as the 22Rv1 cell line was distributed worldwide and, by means that remain to be worked out, into clinical samples from CFS patients.

PREDICTORS OF ACUTE CARDIAC EVENTS IN CORONARY HEART DISEASE PATIENTS WITH AND WITHOUT CHRONIC INFECTION

Free radical oxidation concept considers endothelial damage by oxidized LDL resulting in impaired vasodilation and increased adhesiveness as a key step in atherogenesis. Evidently, correction of the accepted parameters such as dyslipidemia, glycemia, hemostatic indices etc. doesn’t always lead to desirable clinical effects. Therefore, the goal of the study was the identification of plasma lipoperoxyde (LPO) as an additional biomarker influencing course of CAD. 45 patients with stable CAD were randomized into two groups: seronegative (group I) and seroposotive (group II) for antiviral antibodies to Herpes simplex virus I-type (HSV-I), Cytomegalovirus (CMV) and Epstein-Bar virus (EBV). Groups III and IV included 52 seropositive patients without CAD and 20 healthy volunteers. All patients underwent measurements of LPO, plasma lipoprotein profile and CRP. ELISA was used for detection of antiviral antibodies. Intima-medial wall thickness of carotid arteries and abdominal aorta was studied by ultrasound duplex. Positive correlation was established between plasma LPO concentration, LDL level, the titer of specific antiviral antibodies, amount of persisting viruses and acute cardiac events. Increased LPO activity in the seropositive patients indicates that chronic viral infection causes activation of free radical reactions precipitating the development of oxidative stress. In individuals with CAD it can be used as an additional biomarker of expected acute cardiac events.

Undiagnosed hepatitis C virus infection in hemodialysis patients: Value of HCV RNA and liver enzyme levels

At present, routine screening for hepatitis C virus (HCV) infection is based on the detection of antiviral antibodies. Underdiagnosis of HCV infection by using HCV antibody tests, however, still occurs. Additional diagnostic means are provided by the polymerase chain reaction (PCR). The measurement of aminotransferase (ASAT and ALAT) has served as an auxiliary, less specific test. The present research aimed to design practical and low cost strategies to diminish underdiagnosis of HCV infection in dialysis patients. With this purpose in mind, we examined whether aminotransferases values in HCV antibody-negative patients could be related to undiagnosed HCV infection, by using HCV RNA testing by PCR as the gold standard. In 112 hemodialysis patients, we found 78 negative and 34 positive for HCV antibodies. A major finding was that 222 (28.2%) out of the 78 HCV antibodies-negative patients had positive HCV RNA by PCR. In repeated samples taken at six months follow-up from 19 out of these 22 patients, only one of them was positive for anti-HCV antibodies; moreover, a positive HCV RNA by PCR was confirmed in 13 (68.5%) of them. Within the HCV antibody-negative group, the mean values of ASAT, ALAT and gammaglutamiltransferase were higher (P < 0.001, P < 0.001 and P < 0.02, respectively) in the HCV PCR-positive versus the HCV PCR-negative patients. No significant differences were found in the liver enzyme values between the HCV antibody-negative, HCV RNA positive and the HCV antibody positive, HCV RNA positive individuals. Histological samples from two HCV RNA positive, HCV antibody-negative patients disclosed the presence of a mild liver disease. In conclusion, the present study demonstrates the critical importance of HCV RNA determination by PCR in hemodialysis patients who have no detectable circulating antibodies against the HCV. Furthermore, in conditions in which PCR technology is not readily available, we have established that the existence of a moderate increase of aminotransferases is a helpful clue to detect patients with absent HCV antibodies, and might represent an useful, low cost tool for HCV screening in dialysis patients.

An ELISA based on whole virus for the detection of antibodies to small-ruminant lentiviruses.

A new ELISA kit was developed, based on highly purified whole-virus antigen derived from the Swiss maedi-visna virus strain OLV. The sensitivity, specificity and accuracy of this assay were compared with that of an established ELISA based on recombinant GAG (group-specific antigens)-GST (glutathione S-transferase) fusion protein expressed in E. coli (GAG-GST ELISA). The whole-virus ELISA exhibits at least comparable specificity (99.3%) but higher sensitivity (98.6 versus 86.3%) and agreement with the 'true' status beyond chance in the detection of antiviral antibodies in serum from goats. Antibodies in milk samples are detected with higher specificity (98.9 versus 97.8%) but lower sensitivity (91.4 versus 98.2%) than in GAG-GST ELISA. The specificity of the new ELISA in the detection of antibodies in serum might be superior, since a set of 40 samples falsely rated positive in GAG-GST ELISA in routine diagnostic work was negative in the new ELISA. In both assays, milk samples can be tested instead of serum, although with slightly reduced sensitivity in the new ELISA. The major advantage of the new test kit is the low number of equivocal samples needing confirmation in a supplementary test. Results obtained with sheep sera indicate that the new ELISA kit is also suitable for the detection of antibodies to maedi-visna virus.

Expression in Escherichia coli and sequencing of the coding region for the capsid protein of Dutch maedi-visna virus strain ZZV 1050: application of recombinant protein in enzyme-linked immunosorbent assay for the detection of caprine and ovine lentiviruses.

Maedi-visna in sheep and caprine arthritis-encephalitis in goats are caused by two closely related and widespread lentiviruses. The infections are characterized by life-long virus persistence and slow induction of antiviral antibodies. The diagnosis is based on the detection of antiviral antibodies. We have used the polymerase chain reaction (PCR) to amplify a part of the gag gene coding for the entire capsid protein and for parts of the matrix and nucleocapsid proteins. Sequencing of the PCR fragment of the Dutch maedi-visna virus strain ZZV 1050 revealed 85 and 92% homology to the DNA and deduced amino acid sequences, respectively, of the distantly related Icelandic visna virus strain 1514. The respective homologies with caprine arthritis-encephalitis virus strain CO were 76 and 80%. The PCR fragment was cloned into pGEX-2T and expressed as a glutathione S-transferase fusion protein. The recombinant protein could be detected on immunoblots by using a monoclonal antibody and polyclonal antisera and was further purified by glutathione-based affinity chromatography. Enzyme-linked immunosorbent assay with purified recombinant fusion protein is shown to be a sensitive and specific diagnostic tool for the detection of lentiviral infection in goats and sheep.

Expression of Human Parvovirus B19 Structural Protein in E. coli and Detection of Antiviral Antibodies in Human Serum

Human Parvovirus (B19), a small single stranded DNA virus, has been associated with a variety of different human diseases including aplastic crisis and erythema infectiosum. To date there is no practical source of viral antigen for screening potentially infected patients. Starting with previously isolated genomic clones and using a combination of in vitro and in vivo techniques, the major coding region of the B19 virion protein structural gene was expressed in E. coli as a 196 kD β-galactosidase fusion protein. Using this recombinant antigen, antibodies to B19 from the sera of ten patients known to be infected with the virus were detected by Western blot analysis. The sera from 50 normal healthy individuals were also tested and 44% of the subjects showed a positive immunoreaction to the B19-β-galactosidase fusion protein.

Detection of Antiviral Antibodies with Predetermined Specificity Using Synthetic Peptide-beta-Lactamase Conjugates: Application to Antibodies Specific for the preS Region of the Hepatitis B Virus Envelope Proteins

Amino acid sequences coded for by the preS region of the hepatitis B virus (HBV) envelope gene are present both in HBV and in subviral hepatitis B surface antigen (HBsAg) particles. Consequently, anti-preS-specific antibodies are elicited during the course of HBV infection. Such antibodies are virus-neutralizing. Therefore, it is important to determine whether or not vaccination with HBsAg also induces an anti-preS-specific immune response. We describe here an enzyme-linked immunosorbent assay applicable for the screening of sera from vaccinated individuals for anti-preS antibodies. IgG from serum specimens was adsorbed to staphylococcal Protein A on a superparamagnetic support and subsequently mixed with a synthetic peptide analogue [preS(120-145)] covalently linked to beta-lactamase. The presence of anti-preS in serum specimens resulted in binding of the conjugated beta-lactamase to the magnetic support. The adsorbed enzyme was quantified colorimetrically.