Highly drug/target-tolerant neutralizing antibody (NAb) assay development through target-based drug depletion and drug-based NAb extraction for an anti-EGFR therapeutic monoclonal antibody.

The reduction of immunogenicity is fundamental for the development of biobetter Erbitux, given that the development of an immune response reduces treatment efficacy and may lead to potential side effects. One of the requirements for the clinical research of a Erbitux biobetter candidate (CMAB009) is to develop a neutralizing antibody (NAb) assay, and sufficient drug and target tolerance for the assay is necessary. Here, we describe the development of a competitive ligand binding (CLB) assay for CMAB009 with high drug and target tolerance through target-based drug depletion and drug-based NAb extraction, the integrated experimental strategy was implemented to simultaneously mitigate drug interference and enhance target tolerance. Following troubleshooting and optimization, the NAb assay was validated for clinical sample analysis with the sensitivity of 92 ng/mL, drug tolerance of 70 µg/mL and target tolerance of 798 ng/mL. The innovative drug depletion and NAb extraction achieved though the combination of drug and target beads would enable the development of reliable NAb assays for many other therapeutics that overcome drug and its target interference for more precise and sensitive NAb assessment.

Application and Validation of SARS-CoV-2 RBD Neutralizing ELISA Assay.

The establishment of an approach for detecting the anti-severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-receptor-binding domain (RBD) neutralizing antibodies (nAbs) by a safe, easy, and rapid technique without requiring the use of live viruses is essential for facing the coronavirus disease 2019 (COVID-19) pandemic. Depending on competitive enzyme-linked immunosorbent assay (ELISA) methodology, the current study assay was designed to simulate the virus-host interaction using purified SARS-COV-2-RBD from the spike protein and the host cell receptor human angiotensin-converting enzyme 2 protein. The performance of this in-house neutralizing ELISA assay was validated using freshly prepared standards with different known concentrations of the assay. In this regard, a cohort of 50 serum samples from convalescent COVID-19 individuals with different disease severity at different time points post-recovery and a cohort of 50 serum samples from healthy individuals were processed by the in-house developed assay for detecting SARS-CoV-2 nAbs, in comparison with a commercial total anti-SARS-CoV-2 IgG antibody assay as a gold standard. The assay obtained a sensitivity of 88% (95% CI: 75.69-95.47) and a specificity of 92% (95% CI: 80.77- 97.78%). A negative strong correlation was demonstrated in the standard curve between the optical density absorbance and log concentration of the nAbs with a statistical measure of r2 (coefficient of determination) = 0.9539. The SARS-COV-2-RBD neutralizing ELISA assay serves as a high throughput qualitative and quantitative tool that can be applied in most laboratory settings without special biosafety requirements to detect anti-RBD nAbs for seroprevalence, pre-clinical, and clinical evaluation of COVID-19 vaccines efficiency and the rapid selection of convalescent plasma donors for the treatment of COVID-19 patients.

An automated high-throughput enterovirus D68 microneutralization assay platform.

Virus neutralization assays, widely used to detect and quantify antibodies induced by virus infection, are considered the gold standard for enterovirus serology testing. Conventional microneutralization assays have been used to assess enterovirus D68 (EV-D68) seroprevalence. While manual or automated 96-well assays are valuable, higher-density assays that increase throughput provide the opportunity to more efficiently screen large, population-based serology collections, as well as to test sample sets against multiple virus strains on the same plate or within the same run. Here, automation was implemented for bulk reagent dispensing, serial dilutions, and luminescence measurement to develop a 384-well enterovirus microneutralization assay that increases overall testing throughput, maintains the reproducibility of the standard 96-well assay, and reduces sample volume usage. EV-D68 strains Fermon, 14-18953, and 18-23087 were used to evaluate the automated 384-well microneutralization assay and compare to the conventional 96-well assay. Sensitivity and specificity were evaluated using pooled human sera and positive and negative control antisera. The Lower Limit of quantitation (LLOQ) was the same as for the 96-well assay and coefficients of variations (CV) of 7.35 %, 5.97 %, and 2.85 % for the three EV-D68 strains respectively, were well below the typical goal of ≤ 20 % CV for accuracy. Z-factor analysis yielded results of 0.694, 0.638, and 0.852, for the three EV-D68 strains respectively, indicating a high level of precision, reliability, and robustness. Intra-assay (7.25 %) and inter-assay (7.12 %) variability were well below 20 % CV. Moreover, the 96-well and 384-well versions of the assay were highly concordant, with a 0.955 correlation coefficient in titers obtained for 50 sera tested. Validation of this automated 384-well microneutralization will support its use in large serology screens assessing the presence of EV-D68 neutralizing antibodies in human populations.

Evaluation of a commercial ELISA as alternative to plaque reduction neutralization test to detect neutralizing antibodies against SARS-CoV-2.

High-throughput detection of neutralizing antibodies against SARS-CoV-2 presents a valuable tool for vaccine trials or investigations of population immunity. We evaluate the performance of the first commercial surrogate virus neutralization test (sVNT, GenScript Biotech) against SARS-CoV-2 plaque reduction neutralization test (PRNT) in convalescent and vaccinated individuals. We compare it to five other ELISAs, two of which are designed to detect neutralizing antibodies. In 491 pre-vaccination serum samples, sVNT missed 23.6% of PRNT-positive samples when using the manufacturer-recommended cutoff of 30% binding inhibition. Introducing an equivocal area between 15 and 35% maximized sensitivity and specificity against PRNT to 72.8-93.1% and 73.5-97.6%, respectively. The overall diagnostic performance of the other ELISAs for neutralizing antibodies was below that of sVNT. Vaccinated individuals exhibited higher antibody titers by PRNT (median 119.8, IQR 56.7-160) and binding inhibition by sVNT (median 95.7, IQR 88.1-96.8) than convalescent patients (median 49.1, IQR 20-62; median 52.9, IQR 31.2-76.2). GenScript sVNT is suitable to screen for SARS-CoV-2-neutralizing antibodies; however, to obtain accurate results, confirmatory testing by PRNT in a equivocal area is required. This equivocal area may require adaptation for use in vaccinated individuals, due to higher antibody titers.

Neutralizing Antibodies and Cytokines in Breast Milk After Coronavirus Disease 2019 (COVID-19) mRNA Vaccination.

OBJECTIVE: To evaluate immune responses to coronavirus disease 2019 (COVID-19) mRNA-based vaccines present in breast milk and transfer of the immune responses to breastfeeding infants. METHODS: We enrolled 30 lactating women who received mRNA-based COVID-19 vaccines from January through April 2021 in this cohort study. Women provided serial milk samples, including milk expressed before vaccination, across 2-3 weeks after the first dose, and across 3 weeks after the second dose. Women provided their blood, spotted on cards (dried blood spots), 19 days after the first dose and 21 days after the second dose. Stool samples from the breastfed infants were collected 21 days after mothers' second vaccination. Prepandemic samples of milk, dried blood spots, and infant stool were used as controls. Milk, dried blood spots, and infant stool were tested by enzyme-linked immunosorbent assay for receptor-binding domain (RBD)-specific immunoglobulin (Ig)A and IgG. Milk samples were tested for the presence of neutralizing antibodies against the spike and four variants of concern: D614G, Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1). Levels of 10 cytokines were measured in milk samples. RESULTS: Milk from COVID-19-immunized women neutralized the spike and four variants of concern, primarily driven by anti-RBD IgG. The immune response in milk also included significant elevation of interferon-γ. The immune response to maternal vaccination was reflected in breastfed infants: anti-RBD IgG and anti-RBD IgA were detected in 33% and 30% of infant stool samples, respectively. Levels of anti-RBD antibodies in infant stool correlated with maternal vaccine side effects. Median antibody levels against RBD were below the positive cutoffs in prepandemic milk and infant stool samples. CONCLUSION: Humoral and cellular immune responses to mRNA-based COVID-19 vaccination are present in most women's breast milk. The milk anti-RBD antibodies can neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike and variants of concern. Anti-RBD antibodies are transferred to breastfed infants, with the potential to confer passive immunity against SARS-CoV-2.

SARS-CoV-2 Omicron Spike recognition by plasma from individuals receiving BNT162b2 mRNA vaccination with a 16-week interval between doses.

Continuous emergence of SARS-CoV-2 variants of concern (VOCs) is fueling the COVID-19 pandemic. Omicron (B.1.1.529) rapidly spread worldwide. The large number of mutations in its Spike raise concerns about a major antigenic drift that could significantly decrease vaccine efficacy and infection-induced immunity. A long interval between BNT162b2 mRNA doses elicits antibodies that efficiently recognize Spikes from different VOCs. Here, we evaluate the recognition of Omicron Spike by plasma from a cohort of SARS-CoV-2 naive and previously infected individuals who received their BNT162b2 mRNA vaccine 16 weeks apart. Omicron Spike is recognized less efficiently than D614G, Alpha, Beta, Gamma, and Delta Spikes. We compare with plasma activity from participants receiving a short (4 weeks) interval regimen. Plasma from individuals of the long-interval cohort recognize and neutralize better the Omicron Spike compared with those who received a short interval. Whether this difference confers any clinical benefit against Omicron remains unknown.

Neutralizing antibody responses over time in demographically and clinically diverse individuals recovered from SARS-CoV-2 infection in the United States and Peru: A cohort study.

BACKGROUND: People infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) experience a wide range of clinical manifestations, from asymptomatic and mild illness to severe illness and death, influenced by age and a variety of comorbidities. Neutralizing antibodies (nAbs) are thought to be a primary immune defense against the virus. Large, diverse, well-characterized cohorts of convalescent individuals provide standardized values to benchmark nAb responses to past SARS-CoV-2 infection and define potentially protective levels of immunity. METHODS AND FINDINGS: This analysis comprises an observational cohort of 329 HIV-seronegative adults in the United States (n = 167) and Peru (n = 162) convalescing from SARS-CoV-2 infection from May through October 2020. The mean age was 48 years (range 18 to 86), 54% of the cohort overall was Hispanic, and 34% identified as White. nAb titers were measured in serum by SARS-CoV-2.D614G Spike-pseudotyped virus infection of 293T/ACE2 cells. Multiple linear regression was applied to define associations between nAb titers and demographic variables, disease severity and time from infection or disease onset, and comorbidities within and across US and Peruvian cohorts over time. nAb titers peaked 28 to 42 days post-diagnosis and were higher in participants with a history of severe Coronavirus Disease 2019 (COVID-19) (p < 0.001). Diabetes, age >55 years, male sex assigned at birth, and, in some cases, body mass index were also independently associated with higher nAb titers, whereas hypertension was independently associated with lower nAb titers. nAb titers did not differ by race, underlying pulmonary disease or smoking. Two months post-enrollment, nAb ID50 (ID80) titers declined 3.5 (2.8)-fold overall. Study limitations in this observational, convalescent cohort include survivorship bias and missing early viral loads and acute immune responses to correlate with the convalescent responses we observed. CONCLUSIONS: In summary, in our cohort, nAb titers after SARS-CoV-2 infection peaked approximately 1 month post-diagnosis and varied by age, sex assigned at birth, disease severity, and underlying comorbidities. Our data show great heterogeneity in nAb responses among people with recent COVID-19, highlighting the challenges of interpreting natural history studies and gauging responses to vaccines and therapeutics among people with recent infection. Our observations illuminate potential correlations of demographic and clinical characteristics with nAb responses, a key element for protection from COVID-19, thus informing development and implementation of preventative and therapeutic strategies globally. TRIAL REGISTRATION: ClinicalTrials.gov NCT04403880.

The Mucosal and Serological Immune Responses to the Novel Coronavirus (SARS-CoV-2) Vaccines.

Background: Although the serological antibody responses induced by SARS-CoV-2 vaccines are well characterized, little is known about their ability to elicit mucosal immunity. Objectives: This study aims to examine and compare the mucosal and systemic responses of recipients of two different vaccination platforms: mRNA (Comirnaty) and inactivated virus (CoronaVac). Methods: Serial blood and nasal epithelial lining fluid (NELF) samples were collected from the recipients of either Comirnaty or CoronaVac. The plasma and NELF immunoglobulins A and G (IgA and IgG) specific to SARS-CoV-2 S1 protein (S1) and their neutralization effects were quantified. Results: Comirnaty induced nasal S1-specific immunoglobulin responses, which were evident as early as 14 ± 2 days after the first dose. In 64% of the subjects, the neutralizing effects of NELF persisted for at least 50 days. Moreover, 85% of Comirnaty recipients exhibited S1-specific IgA and IgG responses in plasma by 14 ± 2 days after the first dose. By 7 ± 2 days after the booster, all plasma samples possessed S1-specific IgA and IgG responses and were neutralizing. The induction of S1-specific plasma antibodies by CoronaVac was IgG dominant, and 83% of the subjects possessed S1-specific IgG by 7 ± 2 days after the booster, with neutralizing effects. Conclusion: Comirnaty induces S1-specific IgA and IgG responses with neutralizing activity in the nasal mucosa; a similar response is not seen with CoronaVac. Clinical Implication: The presence of a nasal response with mRNA vaccine may provide additional protection compared with inactivated virus vaccine. However, whether such widespread immunological response may produce inadvertent adverse effects in other tissues warrants further investigation.

Function Is More Reliable than Quantity to Follow Up the Humoral Response to the Receptor-Binding Domain of SARS-CoV-2-Spike Protein after Natural Infection or COVID-19 Vaccination.

Both the SARS-CoV-2 pandemic and emergence of variants of concern have highlighted the need for functional antibody assays to monitor the humoral response over time. Antibodies directed against the spike (S) protein of SARS-CoV-2 are an important component of the neutralizing antibody response. In this work, we report that in a subset of patients-despite a decline in total S-specific antibodies-neutralizing antibody titers remain at a similar level for an average of 98 days in longitudinal sampling of a cohort of 59 Hispanic/Latino patients exposed to SARS-CoV-2. Our data suggest that 100% of seroconverting patients make detectable neutralizing antibody responses which can be quantified by a surrogate viral neutralization test. Examination of sera from ten out of the 59 subjects which received mRNA-based vaccination revealed that both IgG titers and neutralizing activity of sera were higher after vaccination compared to a cohort of 21 SARS-CoV-2 naïve subjects. One dose was sufficient for the induction of a neutralizing antibody, but two doses were necessary to reach 100% surrogate virus neutralization in subjects irrespective of previous SARS-CoV-2 natural infection status. Like the pattern observed after natural infection, the total anti-S antibodies titers declined after the second vaccine dose; however, neutralizing activity remained relatively constant for more than 80 days after the first vaccine dose. Furthermore, our data indicates that-compared with mRNA vaccination-natural infection induces a more robust humoral immune response in unexposed subjects. This work is an important contribution to understanding the natural immune response to the novel coronavirus in a population severely impacted by SARS-CoV-2. Furthermore, by comparing the dynamics of the immune response after the natural infection vs. the vaccination, these findings suggest that functional neutralizing antibody tests are more relevant indicators than the presence or absence of binding antibodies.

Saliva-Based ELISAs for Effective SARS-CoV-2 Antibody Monitoring in Vaccinated Individuals.

In March 2020, the World Health Organization (WHO) declared a global health emergency-the coronavirus disease 2019 (COVID-19) pandemic. Since then, the development and implementation of vaccines against the virus amidst emerging cases of re-infection has prompted researchers to work towards understanding how immunity develops and is sustained. Serological testing has been instrumental in monitoring the development and persistence of antibodies against SARS-CoV-2 infection, however inconsistencies in detection have been reported by different methods. As serological testing becomes more commonplace, it is important to establish widespread and repeatable processes for monitoring vaccine efficacy. Therefore, we present enzyme linked immunosorbent assays (ELISAs) compatible for antibody detection in saliva as highly accurate, efficacious, and scalable tools for studying the immune response in individuals vaccinated against SARS-CoV-2.

SARS-CoV-2 mucosal antibody development and persistence and their relation to viral load and COVID-19 symptoms.

Although serological studies have shown that antibodies against SARS-CoV-2 play an important role in protection against (re)infection, the dynamics of mucosal antibodies during primary infection and their potential impact on viral load and the resolution of disease symptoms remain unclear. During the first pandemic wave, we assessed the longitudinal nasal antibody response in index cases with mild COVID-19 and their household contacts. Nasal and serum antibody responses were analysed for up to nine months. Higher nasal receptor binding domain and spike protein-specific antibody levels at study inclusion were associated with lower viral load. Older age was correlated with more frequent COVID-19 related symptoms. Receptor binding domain and spike protein-specific mucosal antibodies were associated with the resolution of systemic, but not respiratory symptoms. Finally, receptor binding domain and spike protein-specific mucosal antibodies remained elevated up to nine months after symptom onset.

Vaccinia virus-based vaccines confer protective immunity against SARS-CoV-2 virus in Syrian hamsters.

COVID-19 in humans is caused by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that belongs to the beta family of coronaviruses. SARS-CoV-2 causes severe respiratory illness in 10-15% of infected individuals and mortality in 2-3%. Vaccines are urgently needed to prevent infection and to contain viral spread. Although several mRNA- and adenovirus-based vaccines are highly effective, their dependence on the "cold chain" transportation makes global vaccination a difficult task. In this context, a stable lyophilized vaccine may present certain advantages. Accordingly, establishing additional vaccine platforms remains vital to tackle SARS-CoV-2 and any future variants that may arise. Vaccinia virus (VACV) has been used to eradicate smallpox disease, and several attenuated viral strains with enhanced safety for human applications have been developed. We have generated two candidate SARS-CoV-2 vaccines based on two vaccinia viral strains, MVA and v-NY, that express full-length SARS-CoV-2 spike protein. Whereas MVA is growth-restricted in mammalian cells, the v-NY strain is replication-competent. We demonstrate that both candidate recombinant vaccines induce high titers of neutralizing antibodies in C57BL/6 mice vaccinated according to prime-boost regimens. Furthermore, our vaccination regimens generated TH1-biased immune responses in mice. Most importantly, prime-boost vaccination of a Syrian hamster infection model with MVA-S and v-NY-S protected the hamsters against SARS-CoV-2 infection, supporting that these two vaccines are promising candidates for future development. Finally, our vaccination regimens generated neutralizing antibodies that partially cross-neutralized SARS-CoV-2 variants of concern.

A bioluminescent and homogeneous SARS-CoV-2 spike RBD and hACE2 interaction assay for antiviral screening and monitoring patient neutralizing antibody levels.

Here we describe a homogeneous bioluminescent immunoassay based on the interaction between Fc-tagged SARS-CoV-2 Spike RBD and human ACE2, and its detection by secondary antibodies labeled with NanoLuc luciferase fragments LgBit and SmBit. The assay utility for the discovery of novel inhibitors was demonstrated with a panel of anti-RBD antibodies, ACE2-derived miniproteins and soluble ACE2. Studying the effect of RBD mutations on ACE2 binding showed that the N501Y mutation increased RBD apparent affinity toward ACE2 tenfold that resulted in escaping inhibition by some anti-RBD antibodies. In contrast, while E484K mutation did not highly change the binding affinity, it still escaped antibody inhibition likely due to changes in the epitope recognized by the antibody. Also, neutralizing antibodies (NAbs) from COVID-19 positive samples from two distinct regions (USA and Brazil) were successfully detected and the results further suggest the persistence of NAbs for at least 6 months post symptom onset. Finally, sera from vaccinated individuals were tested for NAbs and showed varying neutralizing activity after first and second doses, suggesting the assay can be used to assess immunity of vaccinated populations. Our results demonstrate the broad utility and ease of use of this methodology both for drug discovery and clinical research applications.

Immunologic memory to SARS-CoV-2 in convalescent COVID-19 patients at 1 year postinfection.

BACKGROUND: Understanding the complexities of immune memory to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is key to gain insights into the durability of protective immunity against reinfection. OBJECTIVE: We sought to evaluate the immune memory to SARS-CoV-2 in convalescent patients with longer follow-up time. METHODS: SARS-CoV-2-specific humoral and cellular responses were assessed in convalescent patients with coronavirus disease 2019 (COVID-19) at 1 year postinfection. RESULTS: A total of 78 convalescent patients with COVID-19 (26 moderate, 43 severe, and 9 critical) were recruited after 1 year of recovery. The positive rates of both anti-receptor-binding domain and antinucleocapsid antibodies were 100%, whereas we did not observe a statistical difference in antibody levels among different severity groups. Accordingly, the prevalence of neutralizing antibodies (nAbs) reached 93.59% in convalescent patients. Although nAb titers displayed an increasing trend in convalescent patients with increased severity, the difference failed to achieve statistical significance. Notably, there was a significant correlation between nAb titers and anti-receptor-binding domain levels. Interestingly, SARS-CoV-2-specific T cells could be robustly maintained in convalescent patients, and their number was positively correlated with both nAb titers and anti-receptor-binding domain levels. Amplified SARS-CoV-2-specific CD4+ T cells mainly produced a single cytokine, accompanying with increased expression of exhaustion markers including PD-1, Tim-3, TIGIT, CTLA-4, and CD39, while the proportion of multifunctional cells was low. CONCLUSIONS: Robust SARS-CoV-2-specific humoral and cellular responses are maintained in convalescent patients with COVID-19 at 1 year postinfection. However, the dysfunction of SARS-CoV-2-specific CD4+ T cells supports the notion that vaccination is needed in convalescent patients for preventing reinfection.

Zika Virus Antibody Titers Three Years after Confirmed Infection.

BACKGROUND: In 2015-2016, a large Zika virus (ZIKV) outbreak occurred in the Americas. Although the exact ZIKV antibody kinetics after infection are unknown, recent evidence indicates the rapid waning of ZIKV antibodies in humans. Therefore, we aimed to determine the levels of ZIKV antibodies more than three years after a ZIKV infection. METHODS: We performed ZIKV virus neutralization tests (VNT) and a commercial ZIKV non-structural protein 1 (NS1) IgG ELISA in a cohort of 49 participants from Suriname who had a polymerase-chain-reaction-confirmed ZIKV infection more than three years ago. Furthermore, we determined the presence of antibodies against multiple dengue virus (DENV) antigens. RESULTS: The ZIKV seroprevalence in this cohort, assessed with ZIKV VNT and ZIKV NS1 IgG ELISA, was 59.2% and 63.3%, respectively. There was, however, no correlation between these two tests. Furthermore, we did not find evidence of a potential negative influence of DENV immunity on ZIKV antibody titers. CONCLUSIONS: ZIKV seroprevalence, assessed with two commonly used serological tests, was lower than expected in this cohort of participants who had a confirmed previous ZIKV infection. This can have implications for future ZIKV seroprevalence studies and possibly for the duration of immunological protection after a ZIKV infection.

Longitudinal Serological Monitoring of Commercial Broiler Breeders for Fowl Adenoviruses (FAdVs)-Presence of Antibodies Is Linked with Virus Excretion.

Currently, the poultry industry worldwide is facing an emerging trend of fowl adenovirus (FAdV)-associated diseases with a significant economic impact, especially in meat-type chickens. Vertical transmission is an important feature of all FAdVs; hence, preventive measures mostly revolve around breeding stocks. However, knowledge about temporal development of FAdV infections in modern commercial settings is rare or even nonexistent. In the present study, longitudinal monitoring for FAdV was conducted in broiler breeder flocks located in a confined geographical region with intensive poultry production in Iran. For this, the antibody status of birds from 4 to 32 wk of age was monitored with a commercial FAdV-ELISA and virus neutralization test (VNT). In parallel, fecal shedding of FAdV was determined at the peak of egg production with real-time PCR and virus isolation. Overall, the commercial ELISA showed seroconversion of flocks before onset of production. VNT resolved in detail infection patterns of individual serotypes with a primordial FAdV-D (FAdV-2/-11) infection, frequently followed by FAdV-E (FAdV-8a, -8b) superinfection. FAdV-A (FAdV-1) was traced in half of the investigated flocks, while no evidence of infection with FAdV-C (FAdV-4, -10) was noted. Common serological profiles between different houses of the same farm indicate an overarching biosecurity. Serological profiles coupled with virological findings at the peak of egg production indicated that higher antibody levels, determined by ELISA, correlated with lower amounts of viral DNA in fecal excretion. Simultaneously, the number of isolated FAdVs belonging to distinct serotypes declined in accordance with a rise of neutralizing antibodies in birds, underlining the significance of serotype-specific antibodies in the epidemiology of FAdV in breeders. Investigations in breeders were complemented with screening of FAdV-associated diseases in local broilers over a 3-yr period; 26 cases of inclusion body hepatitis with dominant involvement of FAdV-11/FAdV-8b, one outbreak of adenoviral gizzard erosion related to FAdV-1, and no evidence of hepatitis-hydropericardium syndrome suggest that identical serotypes are maintained in the local poultry industry.

Artículo regular­Monitoreo serológico longitudinal en reproductores pesados comerciales para detectar adenovirus del pollo (FAdV): la presencia de anticuerpos está relacionada con la excreción de virus. Actualmente, la industria avícola en todo el mundo enfrenta a una tendencia emergente de enfermedades asociadas con adenovirus del pollo (FAdV) con un impacto económico significativo, especialmente en pollos de engorde. La transmisión vertical es una característica importante de los adenovirus del pollo, por lo que las medidas preventivas giran principalmente en torno a las poblaciones de reproductores. Sin embargo, el conocimiento sobre el desarrollo temporal de las infecciones por adenovirus del pollo en los entornos comerciales modernos es escaso o incluso inexistente. En el presente estudio, se llevó a cabo un seguimiento longitudinal de adenovirus del pollo en parvadas de reproductoras pesadas ubicadas en una región geográfica confinada con producción avícola intensiva en Irán. Para ello, se evaluó el estado de anticuerpos de las aves de 4 a 32 semanas de edad con una prueba comercial de ELISA para adenovirus del pollo y por la prueba de virus neutralización (VNT). En paralelo, se determinó la eliminación fecal de adenovirus del pollo en el pico de producción de huevos mediante un método de PCR en tiempo real y por aislamiento del virus. En general, la prueba de ELISA comercial mostró seroconversión de parvadas antes del inicio de la producción. La prueba de virus neutralización reveló en detalle los patrones de infección de los serotipos individuales con una infección primordialmente por FAdV-D (FAdV-2/-11), seguida frecuentemente por una superinfección por FAdV-E (FAdV-8a, - 8b). Se rastreó FAdV-A (FAdV-1) en la mitad de las parvadas investigadas, mientras que no se observó evidencia de infección por FAdV-C (FAdV-4, -10). Los perfiles serológicos comunes entre las diferentes casetas de la misma granja indican una bioseguridad generalizada. Los perfiles serológicos junto con los hallazgos virológicos en el pico de producción de huevo indicaron que los niveles más altos de anticuerpos, determinados por ELISA, se correlacionaron con cantidades más bajas de ADN viral en la excreción fecal. Simultáneamente, el número de adenovirus de pollo aislados pertenecientes a distintos serotipos disminuyó de acuerdo con un aumento de anticuerpos neutralizantes en aves, lo que subraya la importancia de los anticuerpos específicos de serotipo en la epidemiología del adenovirus del pollo en reproductores. Las investigaciones en reproductoras se complementaron con la detección de enfermedades asociadas a adenovirus en pollos de engorde locales durante un período de 3 años; 26 casos de hepatitis por cuerpos de inclusión con participación dominante de FAdV-11/FAdV-8b, un brote de erosión de molleja adenoviral relacionado con FAdV-1 y ninguna evidencia de síndrome de hepatitis-hidropericardio sugieren que se mantienen serotipos idénticos en la industria avícola local.

Durability of antibody response to vaccination and surrogate neutralization of emerging variants based on SARS-CoV-2 exposure history.

Two-dose messenger RNA vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective in preventing symptomatic COVID-19 infection. However, the durability of protection is not known, nor is the effectiveness against emerging viral variants. Additionally, vaccine responses may differ based on prior SARS-CoV-2 exposure history. To investigate protection against SARS-CoV-2 variants we measured binding and neutralizing antibody responses following both vaccine doses. We document significant declines in antibody levels three months post-vaccination, and reduced neutralization of emerging variants, highlighting the need to identify correlates of clinical protection to inform the timing of and indications for booster vaccination.

A Rapid Assay for SARS-CoV-2 Neutralizing Antibodies That Is Insensitive to Antiretroviral Drugs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike pseudotyped virus (PSV) assays are widely used to measure neutralization titers of sera and of isolated neutralizing Abs (nAbs). PSV neutralization assays are safer than live virus neutralization assays and do not require access to biosafety level 3 laboratories. However, many PSV assays are nevertheless somewhat challenging and require at least 2 d to carry out. In this study, we report a rapid (<30 min), sensitive, cell-free, off-the-shelf, and accurate assay for receptor binding domain nAb detection. Our proximity-based luciferase assay takes advantage of the fact that the most potent SARS-CoV-2 nAbs function by blocking the binding between SARS-CoV-2 and angiotensin-converting enzyme 2. The method was validated using isolated nAbs and sera from spike-immunized animals and patients with coronavirus disease 2019. The method was particularly useful in patients with HIV taking antiretroviral therapies that interfere with the conventional PSV assay. The method provides a cost-effective and point-of-care alternative to evaluate the potency and breadth of the predominant SARS-CoV-2 nAbs elicited by infection or vaccines.