Point-of-Care Diagnostic Biosensors to Monitor Anti-SARS-CoV-2 Neutralizing IgG/sIgA Antibodies and Antioxidant Activity in Saliva.

Monitoring biomarkers is a great way to assess daily physical condition, and using saliva instead of blood samples is more advantageous as the process is simple and allows individuals to test themselves. In the present study, we analyzed the titers of neutralizing antibodies, IgG and secretory IgA (sIgA), in response to the SARS-CoV-2 vaccine, in saliva. A total of 19 saliva and serum samples were collected over a 10-month period 3 weeks after the first vaccine, 8 months after the second vaccine, and 1 month after the third vaccine. The ranges of antibody concentrations post-vaccination were: serum IgG: 81-15,000 U/mL, salivary IgG: 3.4-330 U/mL, and salivary IgA: 58-870 ng/mL. A sharp increase in salivary IgG levels was observed after the second vaccination. sIgA levels also showed an increasing trend. A correlation with trends in serum IgG levels was observed, indicating the possibility of using saliva to routinely assess vaccine efficacy. The electrochemical immunosensor assay developed in this study based on the gold-linked electrochemical immunoassay, and the antioxidant activity measurement based on luminol electrochemiluminescence (ECL), can be performed using portable devices, which would prove useful for individual-based diagnosis using saliva samples.

Heterogeneous SARS-CoV-2-Neutralizing Activities After Infection and Vaccination.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) with different resistance levels to existing immunity have recently emerged. Antibodies that recognize the SARS-CoV-2 spike (S) protein and exhibit neutralizing activities are considered the best correlate of protection and an understanding of humoral immunity is crucial for controlling the pandemic. We thus analyzed such antibodies in individuals recovered from infection in 2020 as well as vaccinees after two doses of an mRNA vaccine. Methods: Neutralizing antibody responses against three SARS-CoV-2 variants (D614G, VOCs Beta and Delta) were determined in serum samples from 54 infected individuals (24 non-hospitalized, 30 hospitalized) and 34 vaccinees shortly after symptom onset or second vaccination, respectively, as well as six months later. In addition, the effect of the S sequence of the infecting strain on neutralization was studied. Results: Non-hospitalized patients had the lowest neutralization titers against all variants, while those of hospitalized patients equaled or exceeded those of vaccinees. Neutralizing activity was lower against the two VOCs and declined significantly in all cohorts after six months. This decrease was more pronounced in hospitalized and vaccinated individuals than in non-hospitalized patients. Of note, the specific neutralizing activity (NT titer/ELISA value ratio) was higher in the infected cohorts than in vaccinees and did not differ between non-hospitalized and hospitalized patients. Patients infected with viral strains carrying mutations in the N-terminal domain of the spike protein were impaired in Beta VOC neutralization. Conclusions: Specific neutralizing activities were higher in infected than in vaccinated individuals, and no difference in the quality of these antibodies was observed between hospitalized and non-hospitalized patients, despite significantly lower titers in the latter group. Additionally, antibody responses of infected individuals showed greater heterogeneity than those of vaccinees, which was associated with mutations in the spike protein of the infecting strain. Overall, our findings yielded novel insights into SARS-CoV-2-specific neutralizing antibodies, evolving differently after virus infection and COVID-19 vaccination, which is an important issue to consider in ongoing vaccine strategy improvements.

Strong Cellular Immune Response, but Not Humoral, against SARS-CoV-2 in Oncohematological Patients with Autologous Stem Cell Transplantation after Natural Infection.

Oncohematological patients show a low immune response against SARS-CoV-2, both to natural infection and after vaccination. Most studies are focused on the analysis of the humoral response; therefore, the information available about the cellular immune response is limited. In this study, we analyzed the humoral and cellular immune responses in nine individuals who received chemotherapy for their oncohematological diseases, as well as consolidation with autologous stem cell transplantation (ASCT), after being naturally infected with SARS-CoV-2. All individuals had asymptomatic or mild COVID-19 and were not vaccinated against SARS-CoV-2. These results were compared with matched healthy individuals who also had mild COVID-19. The humoral response against SARS-CoV-2 was not detected in 6 of 9 oncohematological individuals prior to ASCT. The levels of antibodies and their neutralization capacity decreased after ASCT. Conversely, an enhanced cytotoxic activity against SARS-CoV-2-infected cells was observed after chemotherapy plus ASCT, mostly based on high levels of NK, NKT, and CD8+TCRγδ+ cell populations that were able to produce IFNγ and TNFα. These results highlight the importance of performing analyses not only to evaluate the levels of IgGs against SARS-CoV-2, but also to determine the quality of the cellular immune response developed during the immune reconstitution after ASCT.

Impaired Antibody-Dependent Cellular Cytotoxicity in a Spanish Cohort of Patients With COVID-19 Admitted to the ICU.

SARS-CoV-2 infection causes COVID-19, ranging from mild to critical disease in symptomatic subjects. It is essential to better understand the immunologic responses occurring in patients with the most severe outcomes. In this study, parameters related to the humoral immune response elicited against SARS-CoV-2 were analysed in 61 patients with different presentations of COVID-19 who were recruited in Hospitals and Primary Healthcare Centres in Madrid, Spain, during the first pandemic peak between April and June 2020. Subjects were allocated as mild patients without hospitalization, severe patients hospitalized or critical patients requiring ICU assistance. Critical patients showed significantly enhanced levels of B cells with memory and plasmablast phenotypes, as well as higher levels of antibodies against SARS-CoV-2 with neutralization ability, which were particularly increased in male gender. Despite all this, antibody-dependent cell-mediated cytotoxicity was defective in these individuals. Besides, patients with critical COVID-19 also showed increased IgG levels against herpesvirus such as CMV, EBV, HSV-1 and VZV, as well as detectable CMV and EBV viremia in plasma. Altogether, these results suggest an enhanced but ineffectual immune response in patients with critical COVID-19 that allowed latent herpesvirus reactivation. These findings should be considered during the clinical management of these patients due to the potential contribution to the most severe disease during SARS-CoV-2 infection.

Serological anti-SARS-CoV-2 neutralizing antibodies association to live virus neutralizing test titers in COVID-19 paucisymptomatic/symptomatic patients and vaccinated subjects.

A large number of immunoassays have been developed to detect specific anti-SARS-CoV-2 antibodies; however, not always they are functional to neutralize the virus. The reference test for the anti-spike neutralizing antibodies (nAbs) ability to counteract the viral infection is the virus neutralization test (VNT). Great interest is developing on reliable serological assays allowing antibodies concentration and antibody protective titer correlation. The aim of our study was to detect nAbs serum levels in paucisymptomatic, symptomatic and vaccinated subjects, to find a cut-off value able to protect from virus infection. nAbs serum levels were detected by a competitive automated immunoassay, in association to VNT with the SARS-CoV-2 original and British variant strains. The median nAbs concentrations were: 281.3 BAU/ml for paucisymptomatics; 769.4 BAU/ml for symptomatics; 351.65 BAU/ml for the vaccinated cohort; 983 BAU/ml considering only the second dose vaccinated individuals. The original strain VNT analysis showed 1:80 median neutralization titers in paucisymptomatic and vaccinated subjects; 1:160 in symptomatic patients; 1:160 in the second dose groups. The British variant VNT analysis showed lower neutralization titers in paucisymptomatic and vaccinated groups (1:40); the same titer in symptomatic patients (1:160); the second dose group confirmed the original strain titer (1:160). In conclusion, our data showed optimal correlations with a proportional increase between neutralizing activity and antibody concentration, making nAbs detection a good alternative to virus neutralization assays, difficult to carry out in routine laboratories. Finally, ROC curve analysis established a cut-off of 408.6 BAU/ml to identify subjects with a low risk of infection.

A combined strategy to detect plasma samples reliably with high anti-SARS-CoV-2 neutralizing antibody titers in routine laboratories.

The determination of anti-SARS-CoV-2 neutralizing antibodies (NAbs) is of interest in many respects. High NAb titers, for example, are the most important criterion regarding the effectiveness of convalescent plasma therapy. However, common cell culture-based NAb assays are time-consuming and feasible only in special laboratories. Our data reveal the suitability of a novel ELISA-based surrogate virus neutralization test (sVNT) to easily measure the inhibition-capability of NAbs in the plasma of COVID-19 convalescents. We propose a combined strategy to detect plasma samples with high NAb titers (≥ 1:160) reliably and to, simultaneously, reduce the risk of erroneously identifying low-titer specimens. For this approach, results of the sVNT assay are compared to and combined with those acquired from the Euroimmun anti-SARS-CoV-2 IgG assay. Both assays are appropriate for high-throughput screening in standard BSL-2 laboratories. Our measurements further show a long-lasting humoral immunity of at least 11 months after symptom onset.

Can We Protect Those We Care for in A Pandemic? - Prevalence of Neutralizing Antibodies against SARS-CoV-2 in Nursing Homes.

In December 2019, the People's Republic of China and the World Health Organization first reported on a cluster of pneumonia with an unknown cause. Nine months later more than 1.4 million people have died from COVID 19. In this work, the effects of the COVID 19 pandemic on five nursing homes in Austria, which cared for 889 residents in the first half of 2020, were examined. The research question was whether the measures taken were appropriate to prevent an outbreak within the individual facilities. To detect previously unrecognized infections, the present study evaluated the prevalence of neutralizing antibodies against the SARS-CoV-2 virus in residents and employees of the nursing homes. Following the analysis of blood samples, the prospectively collected data was connected to data from screening examinations and data from contact tracing. The present study demonstrated an overall prevalence of neutralizing antibodies against the SARS-CoV-2 virus in nursing homes of 3.7%. Whereas the prevalence in those facilities that have never been hit by an outbreak is 0%, the prevalence in those facilities with an outbreak is up to 4.9%. Neutralizing antibodies against SARS-CoV-2 were detected in 35 persons. A retrospective analysis of all 5 included nursing homes demonstrated that upon regular clinical screening in combination with PCRs an infection with SARS-COV-2 was detected in 66 residents and 24 employees from different professional groups. In only 25 of the 35 persons with neutralizing antibodies against SARS-CoV-2 an infection was proven in advance. This study suggests that specific measures can prevent transmission within a health care facility. Nevertheless, the results also show that a risk reduction to 0% cannot be achieved. In preparation for further pandemic waves there is still the need to reduce the probability of a transmission in nursing homes with specific test strategies.

Immunogenicity of Adjuvanted Psoralen-Inactivated SARS-CoV-2 Vaccines and SARS-CoV-2 Spike Protein DNA Vaccines in BALB/c Mice.

The development of a safe and effective vaccine to protect against COVID-19 is a global priority due to the current high SARS-CoV-2 infection rate. Currently, there are over 160 SARS-CoV-2 vaccine candidates at the clinical or pre-clinical stages of development. Of these, there are only three whole-virus vaccine candidates produced using ß-propiolactone or formalin inactivation. Here, we prepared a whole-virus SARS-CoV-2 vaccine (SARS-CoV-2 PsIV) using a novel psoralen inactivation method and evaluated its immunogenicity in mice using two different adjuvants, alum and Advax-2. We compared the immunogenicity of SARS-CoV-2 PsIV against SARS-CoV-2 DNA vaccines expressing either full-length or truncated spike proteins. We also compared the psoralen-inactivated vaccine against a DNA prime, psoralen-inactivated vaccine boost regimen. After two doses, the psoralen-inactivated vaccine, when administered with alum or Advax-2 adjuvants, generated a dose-dependent neutralizing antibody responses in mice. Overall, the pattern of cytokine ELISPOT responses to antigen-stimulation observed in this study indicates that SARS-CoV-2 PsIV with the alum adjuvant promotes a Th2-type response, while SARS-CoV-2 PsIV with the Advax-2 adjuvant promotes a Th1-type response.