Corrigendum: XAV-19, a swine glyco-humanized polyclonal antibody against SARS-CoV-2 spike receptor-binding domain, targets multiple epitopes and broadly neutralizes variants.

[This corrects the article DOI: 10.3389/fimmu.2021.761250.].

Detection of RNA-dependent RNA polymerase of porcine epidemic diarrhea virus.

The RNA synthesis of porcine epidemic diarrhea virus (PEDV) is a sophisticated process performed by a multilingual viral replication complex, together with cellular factors. A key enzyme of this replication complex is RNA-dependent RNA polymerase (RdRp). However, there is limited knowledge about PEDV RdRp. In our present study, a polyclonal antibody against RdRp was prepared by using a prokaryotic expression vector pET-28a-RdRp to study the function of PEDV RdRp and provide a tool to investigate PEDV pathogenesis. In addition, the enzyme activity and half-life of PEDV RdRp were investigated. The result showed that the polyclonal antibody against PEDV RdRp was successfully prepared and was able to be used to detect PEDV RdRp by immunofluorescence and western blotting. Additionally, enzyme activity of PEDV RdRp reached nearly 2 pmol/µg/h and the half-life of PEDV RdRp was 5.47 h.

PEGylation Prolongs the Half-Life of Equine Anti-SARS-CoV-2 Specific F(ab')2.

Therapeutic antibodies-F(ab')2 obtained from hyperimmune equine plasma could treat emerging infectious diseases rapidly because of their high neutralization activity and high output. However, the small-sized F(ab')2 is rapidly eliminated by blood circulation. This study explored PEGylation strategies to maximize the half-life of equine anti-SARS-CoV-2 specific F(ab')2. Equine anti-SARS-CoV-2 specific F(ab')2 were combined with 10 KDa MAL-PEG-MAL in optimum conditions. Specifically, there were two strategies: Fab-PEG and Fab-PEG-Fab, F(ab')2 bind to a PEG or two PEG, respectively. A single ion exchange chromatography step accomplished the purification of the products. Finally, the affinity and neutralizing activity was evaluated by ELISA and pseudovirus neutralization assay, and ELISA detected the pharmacokinetic parameters. The results displayed that equine anti-SARS-CoV-2 specific F(ab')2 has high specificity. Furthermore, PEGylation F(ab')2-Fab-PEG-Fab had a longer half-life than specific F(ab')2. The serum half-life of Fab-PEG-Fab, Fab-PEG, and specific F(ab')2 were 71.41 h, 26.73 h, and 38.32 h, respectively. The half-life of Fab-PEG-Fab was approximately two times as long as the specific F(ab')2. Thus far, PEGylated F(ab')2 has been prepared with high safety, high specificity, and a longer half-life, which could be used as a potential treatment for COVID-19.

Il Rischio Di Reinfezione Da Sars-Cov-2 E Le Implicazioni Per La Diagnostica

One of the most interesting aspects of the COVID-19 pandemic is that a variable percentage of patients (from 2% to 69%) could have a repeated positivity following hospital discharge or even several weeks after clinical recovery (1). There are multiple reasons why a positive result to SARS-CoV-2, usually ascertained by RT-PCR, may be detected again, including reinfection, disease reactivation, prolonged viral shedding or false positive results (1-2). Since the beginning of the pandemic, several authors have reported the possibility of reinfection by SARS-CoV-2 or reactivation of a latent infection, calling for urgent attention from researchers, as well as public health policymakers. In our study we estimate incidence rate of 3.5% of reinfection in the Province of Modena in the first 6 months of 2021. Reinfection rates according to vaccinated or non-vaccinated subjects were 0.6% vs 1.1% (p<0.0001). Multiple questions regarding reinfection associated with SARS-CoV-2 are still ongoing. What is the pathophysiological mechanism for reinfection? Who are the subjects with a higher risk of reinfection? What is the clinical burden for reinfected patients? Reinfection with the SARS-CoV-2 virus can be mainly attributed to two phenomena: decay of the immune response and viral mutations that favor the appearance of new variants (3-5). Currently, there are discordant rates of reinfection reported in SRs (ranging from 0-50%), which could partially be explained by the heterogeneous adopted definitions of reinfection. Today, there is still no universal agreement on the determination of the correct time period between positive results for SARS-CoV-2 for the definition of reinfection, although the definition provided by CDC is the most accredited (https://www.cdc.gov/ coronavirus/2019-ncov/php/invest-criteria.html). It has been pointed out that the severity of reinfection depends on the individual immune response, as well as both the viral load and the SARS-CoV-2 variants causing the reinfection. New virus variants could evade immune responses acquired in subjects with infections from previous variants or reduce the capacity for neutralization by polyclonal antibodies (4). This issue suggests the need to increase the current knowledge about the degree of protection provided against SARS-CoV-2, leading the development of vaccines and the creation and implementation of appropriate interventional strategies. Because COVID-19 is a relatively new disease, several aspects of its progression and long-term health effects are unknown, one of the aspects that have become more relevant as time goes by is the impact that reinfections. There is a real, albeit rare risk of SARS-CoV-2 reinfection. Nevertheless, a standardized approach to identify and report reinfection cases should be developed.

Analysis of S protein cross-interference in three different corona virus strains

Objective: To investigate the S protein cross-interference of the HB02 coronavirus strain, the Delta coronavirus strain and the Omicron coronavirus strain using the developed method of polyclonal enzyme-linked immunoassay (ELISA). Method(s): The monovalent vaccines of HB02 strain, Delta strain and Omicron strain were prepared in different ratios and combinations. The inactivated vaccines of HB02 strain, Delta strain and Omicon strain (Vero cell) S protein antigen ELISA method was used for detection, and the detection results were compared and the interference rate was calculated. At the same time, the interference rates between the monovalent antigen detection results of the three SARS-CoV-2 strains were analyzed. Result(s): The results of the two strains at different doses showed that the interference rate ranged from 4.21% to 29.53% when Delta strain was added to HB02 strain along with the increasing dose. When Omicron strain was increasingly added to HB02 strian, the interference rate ranged 4.40%~12.75%, and when HB02 strain was increasingly added to Delta strain, the interference rate ranged 56.17%~464.21%. The interference rate ranged from 6.29% to 25.91% when Omicron strain was added to Delta strain from a low to high dose. When HB02 strain was added to Omicron strain, the interference rate ranged from 32.05% to 337.22%. When Delta strain was added to Omicron strain, the interference rate ranged from 44.91% to 252.14%. The results of different doses of the three strains showed that the interference rate ranged from 2.79% to 22.86% when different doses of Delta strain+Omicron strain were added to HB02 strain. When different doses of HB02+Omicron strains were added to Delta strain, the interference rate was 67.16% to 267.95%. When different doses of HB02+Delta strains were added to Omicron strain, the interference rate was 100.66%~412.54%. In addition, the interference rates of monovalent Delta strain and Omicron strain detected by HB02 antigen detection kit were 70% and 90%, respectively. The interference rates of Delta strain antigen detection kit for monovalent HB02 strain and Omicron strain were 30% and 35%, respectively. The interference rates of Omicron strain antigen detection kit for monovalent HB02 strain and Delta strain were 5% and 10% respectively. Conclusion(s): Due to the high similarity of S protein in the HB02, Delta and Omicron COVID-19 vaccines, different strains were mixed at series ratios, and different components had different degrees of interference. When the components of the two strains were mixed at different doses, both Delta strain and Omicron strain showed positive interference to the HB02 strain, and the Delta strain showed a gradually increasing trend with the increasing Delta strain doses, while the interference rate did not change significantly with the variation of Omicron strain dose. HB02 strain showed strong positive interference to Delta strain, and the interference rate gradually increased with the increase of HB02 strain, while Omicron strain showed weak positive interference to Delta strain, and the interference rate did not change significantly with the increase of Omicron strain dose. Both HB02 strain and Delta strain showed strong positive interference to Omicron strain, and showed a stepwise increasing trend with the increase of the composition ratio of HB02 strain or Delta strain. When the three strains were mixed at different ratios, the Delta strain+Omicron strain showed positive interference to the HB02 strain, but the interference rate did not change significantly with the increase of the composition of Delta strain+Omicron strain. HB02 strain+Omicron strain showed strong positive interference to Delta strain. However, when the composition of HB02 strain and Delta strain was unchanged, the interference of Omicron strain to Delta strain did not change significantly no matter how Omicron strain changed. The results showed that HB02 strain had strong positive interference to Delta strain, but Omicron strain did not exert obvious interference to Delta strain. HB02+D lta strain showed strong positive interference to Omicron strain, and the ratio of HB02 and Delta strain increased stepwise. Different COVID-19 antigen detection kits were used to detect the monovalent COVID-19 samples of the three virus strains. Among them, HB02 antigen detection kit had the highest interference rate to Omicron strain samples, but Omicron strain antigen detection kit could effectively detect HB02 COVID-19 antigen. The Delta strain antigen detection kit could detect some COVID-19 antigens of HB02 strain and Omicron strain. Copyright © 2022, Chinese Journal of New Drugs Co. Ltd. All right reserved.

Preparation of the RIPK3 Polyclonal Antibody and Its Application in Immunoassays of Nephropathogenic Infectious Bronchitis Virus-Infected Chickens.

Receptor interacting protein kinase 3 (RIPK3) is a vital serine/threonine kinase in regulating the programmed destruction of infected cells to defend against RNA viruses. Although the role of RIPK3 in viruses in mice is well characterized, it remains unclear where in nephropathogenic infectious bronchitis virus (NIBV) in chickens. Here, we use a self-prepared polyclonal antibody to clarify the abundance of RIPK3 in tissues and define the contributions of RIPK3 in tissue damage caused by NIBV infection in chickens. Western blot analyses showed that RIPK3 polyclonal antibody can specifically recognize RIPK3 in the vital tissues of Hy-Line brown chicks and RIPK3 protein is abundantly expressed in the liver and kidney. Moreover, NIBV significantly upregulated the expression levels of RIPK3 in the trachea and kidney of chicks in a time-dependent manner. In addition, the activation of necroptosis in response to NIBV infection was demonstrated by the coimmunoprecipitation (CoIP) experiments through RIPK3 in the necrosome, which phosphorylates its downstream mixed-spectrum kinase structural domain-like protein (MLKL). Our findings offered preliminary insights into the key role of RIPK3 protein in studying the underlying mechanism of organ failure caused by NIBV infection.

THE ROLE of MOLECULAR MIMICRY in SARS-COV-2 RELATED AUTOIMMUNE RHEUMATIC DISEASES

Background: Several studies suggested that SARS-CoV-2 infection may induce autoantibodies related to autoimmune rheumatic diseases (ARD). Objectives: To determine whether polyclonal antibodies from SARS-CoV-2 unin-fected patients with ARDs cross-react with SARS-CoV-2 and vice versa. Methods: 90 sera positive at high-titres for 23 common autoantibodies (all sera stored before 2018), were tested for reactivity against proteins of SARS-CoV-2 (spike protein S1, nucleocapsid NC etc) by ELISA and CMIA. Vice versa, 10 monoclonal antibodies against S1 protein (most of them against RBD) were tested for autoantibody reactivity by indirect immunofuorescence, ELISA, immunoblot and dot/line immunoassays coated with different antigens. Ten post-COVID sera with high titers of anti-Spike abs were tested by ELISAs for reactivity against various autoantigens related to ARDs. Results: 88 out of 90 samples (%), were totally unreactive to SARS-CoV-2 proteins;2 sera, one anti-CCP and one anti-CENP reacted against S protein. All sera tested negative for neutralized abs against SARS-CoV-2. None of 10 sera from SARS-CoV-2 infected patients reacted with different autoantigens by molecular assays. None of the 10 monoclonal abs against S1 protein reacted with 23 different self-antigens. On HEp2 cells as substrate for IIF, 3 of the 10 monoclonal abs gave a low-titre coarse speckled pattern. No reactivity was found by IIFL using tissue substrates. Conclusion: Our data do not suggest a dominant role for molecular mimicry and immunological cross reactivity as a trigger of autoantibodies related to ARDs.

Characteristics of patients with hematologic malignancies without seroconversion post-COVID19 third vaccine dosing (3V): Real-world data from large midwestern healthcare system

Background: Patients (Pts) with hematologic malignancies (HM) are at greater risk of severe morbidity and mortality caused by COVID19 and show a lower response to the two-dose COVID19 mRNA vaccine series. The primary vaccine series now includes a third dose of the COVID19 vaccine (3V) for immunocompromised Pts. The objective of this study was to explore the characteristics of HM patients who had no change in SARS-CoV-2 spike protein titer levels post 3V (-/-) to gain a better understanding of the drivers of serostatus. Methods: This retrospective cohort study analyzed Pt data on SARS-CoV-2 spike IgG antibody titers pre- and post- 3V across the healthcare system. This study included 268 fully vaccinated HM Pts diagnosed with HM between October 31, 2019 and January 31, 2022 and had a negative serostatus prior to 3V. Post 3V titers were obtained 21 days after 3V. Demographics, association between characteristics and seroconversion status, and odds ratios were all assessed (table). Results: Pts with Non-Hodgkin lymphoma (NHL) had 6 times the odds of not seroconverting compared to multiple myeloma (MM) (CI 1.88 - 19.12, P = .0010). NHL also have about 14 times the odds of not seroconverting compared to Pts diagnosed with other HM conditions, which included: neoplasms of uncertain behavior and disorders of white blood cells (CI 1.72 - 112.44, P = .0021). 90% of seronegative Pts showed no spike IgG antibody reaction to 3V as indicated by pre- and post- 3V index values. Demographics, previous COVID19 infection, and vaccine type were not significantly associated with seroconversion. Conclusions: HM patients who are not seroconverting after 3V, suggest a prioritized population for continued increased behavioral precautions, additional vaccination efforts, including a fourth dose of an mRNA COVID19 vaccine, as well as passive immunity boosting through monoclonal and polyclonal antibodies.

LLAMA (Lama glama) ANTISERUM AGAINST SARS-CoV-2

The receptor-binding domain (RBD) of SARS-CoV-2 Spike protein constitutes the key access for the virus inside the host cell. A positive correlation between titers of anti-RBD immunoglobulin G and antibodies (Ab) capable of neutralizing the virus has been demonstrated. In this context, passive immunotherapies such as convalescent plasma and hyperimmune equine serum have gained relevance as therapies against COVID-19. Another promising alternative is the use of polyclonal Ab from llamas (Lama glama), because of their unique features. For this reason, we aimed to obtain recombinant RBD as an immunogen to generate anti-RBD Ab in llamas. To achieve this, HEK 293 cells were transfected and transduced with the RDB encoding sequence, resulting in higher yields with this last method. The RBD was purified by affinity chromatography. An immunization schedule was designed and evaluated on two male animals, which were initially inoculated with RBD, followed by periodical boosters. Exploratory bleedings were performed in order to evaluate the reached titers, and larger bleedings in order to obtain enriched plasma with anti-RBD Ac. Ac quantification was accomplished by an in-house ELISA. Results showed that the immunization scheme was successful, achieving a maximum titer of 168000 at 28 days post-immunization. The results lay the foundations for the production of polyclonal anti-RBD Ab.

PURIFICATION OF ANTI-RBD ANTIBODIES OBTAINED FROM LLAMA (Lama glama) FOR POTENTIAL COVID-19 THERAPY

Despite worldwide efforts to develop different treatments for SARS-CoV-2 COVID-19, the situation remains critical, requiring rapid and effective strategies. In this regard, antibodies (Ab) have demonstrated clinical potential. Among them, camelid nanoAb (VHH) arise as a possible alternative, as they recognize epitopes which are inaccessible to conventional Ab. Further advantages of VHH are their small size, high solubility, high stability, and resistance to low pH. The aim of this work is to describe a purification scheme of different isotypes of anti-SARS-CoV-2 immunoglobulin G (IgG) produced after immunizing two llamas (Lama glama). To achieve this, plasma was injected into an affinity chromatographic column (Protein G), and the resulting fractions were analyzed by SDS-PAGE under non-reducing conditions. The anti-RBD titers were determined by an “in house” ELISA, reaching titers of 52000 and 13000 for IgG1 and IgG3 fractions, respectively. Subsequently, an affinity column (HiTrap NHS-activated) was prepared to separate monospecific anti-RBD polyclonal Ab. RBD produced in our laboratory was covalently coupled to this column, achieving a coupling efficiency of 97%. Different isotypes of monospecific anti-RBD Ab (IgG1: 140 kDa and IgG3: 95 kDa) were obtained. IgG3 represent the starting point for obtaining VHH and/or evaluating their potential use as a therapeutic or preventive alternative, which represents a notable regional contribution in the fight against COVID-19.

Comparison of electrochemical immunosensor platforms for indiscriminate SARS-CoV-2 variant detection

Since the first days of the pandemic, diagnosis of patients infected with the SARS-CoV-2 has been one of the most important parameters to control the virus. For this reason, many studies have been carried out to develop various methods for rapid and accurate diagnosis, but mutations and the occurrence of successive variants have made accurate diagnosis difficult. In this study, a screen-printed carbon electrode was used to develop magnetic nanoparticle (MNP)-based electrochemical biosensing systems that selectively detect SARS-CoV-2 virus and its variants (original, alpha, beta, and delta) in nasopharyngeal swabs. These electrodes were modified with MNPs conjugated to SARS-CoV-2 S1, S2 proteins and swab samples. Then, commercially available SARS-CoV-2-specific anti-S1 and anti-S2 antibodies and antibody cocktails purified from serum samples were applied to the surface and the performance of the platforms was compared. Analytical parameters and electrode surface characterizations were performed by electrochemical measurements after each modification step. After optimization studies of the developed biosensor platforms, the detection of limit for the antibody cocktail- based sensors were determined to be 0.53-0.75 ng/mL, while it was calculated to be 0.93-0.99 ng/mL for the anti-S1 and anti- S2-based sensors. The performance of the platforms in real nasopharyngeal swab samples (negative, original, alpha, beta, and delta variants) was evaluated and it was found that the polyclonal antibody cocktail outperformed the commercial anti-S1 and anti-S2 antibodies. As a result, polyclonal antibody cocktail, with an overall sensitivity, specificity, and accuracy of 100%, is a versatile electrochemical biosensor system for the detection of the different variants of SARS-CoV-2. We hope that the biosensor platform modified with polyclonal antibodies can be used as a potential diagnostic tool that can be applied to such epidemics in the future. Synthetic biology.

STRAIN-SPECIFIC SEROLOGICAL RESPONSE FOLLOWING SARS-CoV-2 VOC INFECTION

Background: The emergence of new SARS-CoV-2 variants raises concerns whether preexisting artificial (vaccine-induced) and natural immunity from prior COVID-19 prevents re-infections. Here, we investigated the differences in primary humoral immune response following SARS-CoV-2 variants of concern (VOCs) infection and aimed to identify the key mutations involved in these differences. Methods: Patients with primary PCR-proven SARS-CoV-2 infection with no history of previous COVID-19 vaccination were included between October 2020 and May 2021 at Amsterdam UMC and via the Dutch SARS-CoV-2 sequence surveillance program. Serum was collected 4-8 weeks after symptom onset and tested for IgG binding and pseudovirus neutralization of the wild-type (WT, Wuhan/D614G), Alpha, Beta and Delta variants. Results: We included 51 COVID-19 patients, who were infected with the WT (n=20), Alpha (n=10), Beta (n=9) or Delta variant (n=12). Generally, the highest neutralization titers were against the autologous virus. After stratifying for hospitalization status, non-hospitalized patients infected with the WT (ID50 817) or Alpha (ID50 2524) variant showed the strongest geometric mean autologous neutralization, followed by the Delta variant (ID50 704) infected participants. By contrast, only one participant infected with the Beta variant showed strong autologous neutralization (median ID50 171). The VOCs also differed in their ability to induce cross-neutralizing responses, with WT-infected patients showing the broadest immune response, followed by Alpha, Delta and Beta infected participants. Additionally, participants infected with the WT, Alpha or Delta variant showed the lowest cross-neutralization against the Beta variant, with a median 5.0-fold (2 to 16-fold), 7.7-fold (2 to 32-fold), and 5.3-fold (1 to 19-fold) reduction compared to the autologous neutralization, respectively. We identified the E484K mutation as the key mutation responsible for this low cross-neutralization. Conclusion: We demonstrated that even small differences in the S protein influences the polyclonal antibody response following infection. The low level of (cross-)neutralization induced by the Beta variant may implicate a higher re-infection risk, but further research of the memory B cell compartment and clinical studies are needed. The broadest cross-neutralizing response observed for WT-infected patients suggests that artificial immunity induced by the current approved COVID-19 vaccines already protects against many re-infections.

PHASE-2 STUDY of SAB-185, A POLYCLONAL ANTIBODY TREATMENT for COVID-19 in ACTIV-2

Background: The discovery and development of SARS-CoV-2 therapies remains a priority. SAB-185 is a Transchromosomic, bovine-derived, fully human polyclonal immunoglobulin product for SARS-CoV-2 being studied in ACTIV-2, randomized controlled platform trial evaluating the safety and efficacy of investigational agents for non-hospitalized adults with mild-moderate COVID-19 Methods: This Phase II trial was a superiority comparison of SAB-185 vs. placebo. Participants with confirmed SAR-CoV-2 infection received intravenous infusion of SAB-185 (3,840 Units/kg) or placebo. Primary outcome measures were proportion of participants with SARS-CoV-2 RNA < lower limit of quantification (LLoQ) in nasopharyngeal (NP) swab, time to improvement in targeted symptoms for 2 consecutive days after Day 0, and safety through Day 28. Secondary outcomes included quantitative NP RNA levels and all-cause hospitalizations and deaths. Antiviral or clinical efficacy and safety criteria for graduation to Phase III were pre-specified. Results: From April to August 2021, randomized participants from 42 sites in the US received SAB-185 (N=107) or placebo (N=106). Median age was 38 years (quartiles: 30,48), 54% female, >98% cis-gender, 7% Black/African-American, 50% Hispanic, and 11% were classified as high-risk for COVID-19 progression, with median 4 days (3,6) from symptom onset. Day 0 NP SARS-CoV-2 RNA levels were similar between SAB-185 and placebo: 4.80 vs 4.80 log10 copies/ml. No differences were observed in the proportion with NP SARS-CoV-2 RNA< lower limit of quantification (LLoQ) in nasopharyngeal (NP) swab, time to improvement in targeted symptoms for 2 consecutive days after Day 0, and safety through Day 28. Secondary outcomes included quantitative NP RNA levels and all-cause hospitalizations and deaths. Antiviral or clinical efficacy and safety criteria for graduation to phase 3 were pre-specified. Conclusion: SAB-185 was safe in this Phase II study. While no significant differences to placebo were seen in symptom duration and proportion of participants with NP SARS-CoV-2 RNA< lower limit of quantification (LLoQ) in nasopharyngeal (NP) swab, time to improvement in targeted symptoms for 2 consecutive days after Day 0, and safety through Day 28. Secondary outcomes included quantitative NP RNA levels and all-cause hospitalizations and deaths. Antiviral or clinical efficacy and safety criteria for graduation to phase 3 were pre-specified.

INCREASE in FALSE POSITIVE 4TH GENERATION HIV TESTS in PATIENTS with COVID-19 DISEASE

Background: A variety of infections and inflammatory conditions have been associated with false positive (FP) serological tests, including those for HIV. In the context of an HIV counseling, testing, and referral program, an apparent increase in FP 4th generation HIV tests was observed among persons infected with SARS-CoV-2. We sought to determine if there was an association of active coronavirus disease-2019 (COVID-19) with a FP HIV test. Methods: This was a retrospective, cross-sectional study from March 2020 to August 2021 at Henry Ford Hospital. Through electronic medical record extraction, all results for SARS-CoV-2 by PCR within + two weeks of a diagnostic HIV 4th generation assay (Elecsys HIV Duo, Roche Diagnostics, Indianapolis, IN) were selected. Confirmatory HIV-1 and HIV-2 antibodies, as well as quantitative HIV RNA, was performed for all positive 4th generation tests. All positive HIV 4th generation assays were independently reviewed and divided into groups of FP, true positives (TP), and true negatives (TN). Variables included age, race, ethnicity, and sex. Statistical analysis was performed in a pairwise fashion using a Chi-squared test. Multivariate logistic regression was used to predict positive COVID-19 tests. Results: A total of 23,278 medical records meeting the above criteria were reviewed. The rates of COVID positive tests were then arranged in groups of HIV TP, FP, and TN. In total, 23,041 patients had a TN HIV test result, 167 patients had a TP, and 70 patients had a FP (Table 1). Those with HIV FP tests had the highest percentage of COVID positive test results at 22.9% (p=0.001), which was significantly higher than HIV TN (10.2%;p=0.197) and HIV TP (7.2%;p=0.001). After adjustment for all covariates, only FP HIV was significantly associated with COVID-19 (OR=7.04;p=0.001). Conclusion: This study reveals that patients with active COVID-19 disease are significantly more likely to have a false positive 4th generation HIV test. The mechanism for this is unknown but may reflect broad polyclonal antibody generation in acute infections or cross-reactivity to antibodies with the SARS-CoV-2 spike protein. Although only a single 4th generation test was evaluated in this study, acute COVID-19 infection should be considered as a potential etiology for a false positive 4th generation HIV test.

PURIFICATION of SPECIFIC ANTIBODIES AGAINST SARS-COV-2 PROTEIN NP USING the FasTAG® SYSTEM

The SARS-CoV-2 coronavirus, which causes respiratory syndrome COVID-19, has a protein nucleocapsid that envelops the viral ssRNA. The main protein of the nucleocapsid is the Np protein, which presents limited homology with nucleoproteins of other coronaviruses and therefore turns out to be an attractive antigen for the development of specific anti-Np antibodies. These antibodies can be used for the development of diagnostic systems that allow the detection of the viral antigen in infected individuals from saliva samples. In this context, our group has developed a labelling system called FasTAG®, which allows the immobilization of recombinant proteins on the surface of Gram+ formaldehyde inactivated bacteria. In this system, the recombinant proteins expressed in heterologous systems are fused to the C-terminal domain of S-Layer proteins of Lactobacillus sp. Then, the intrinsic affinity this domain possesses for the membranes of Gram+ bacteria is used for the immobilization of the recombinant proteins of interest. In this way, it is possible to purify specific antibodies against an antigen of interest. Based on the above, the objective of this work was to evaluate the functionality of the FasTAG® system to purify specific anti-Np antibodies. For this, the recombinant protein Np-FasTAG® was incubated for 12 hours at 4 °C with a matrix made up of B. subtilis inactivated with 3% formaldehyde. Next, for the optimization of the protein fixation process to the matrix, a compound factorial design was carried out, the variables of which were: formaldehyde concentration (0.5-1.5-2.5% v/v) and time of incubation (15-30-45 minutes). The optimal condition was determined as the one that minimizes the detachment of the Np protein and maximizes the detachment of the specific antibodies. Turning out to be the optimal condition for the elaboration of the affinity matrix 2.5% v/v of formaldehyde and 15 minutes. Then, in order to evaluate the application of the affinity matrix in the purification of specific antibodies, it was incubated for 1 hour with polyclonal antibodies obtained from chicken egg yolks and the serum of goats immunized with the Np antigen. Next, to study the elution conditions of the antibodies, a compound factorial design was performed using variables: pH, time, and SDS concentration. The best elution condition was obtained for pH 10.5 and 15 minutes. Subsequently, the purified antibodies were evaluated by SDS-PAGE and ELISA. As a result, it was possible to purify 3.5 μg of anti-Np IgG and 3.1 μg of anti-Np IgY per mg of resin. Finally, the set of experiments carried out here demonstrate the potential and functionality of this system for the purification of specific anti-Np antibodies and their use for diagnostic purposes.

RBD-SPECIFIC POLYCLONAL F(ab)2 FRAGMENTS of EQUINE ANTIBODIES in PATIENTS with MODERATE to SEVERE COVID-19 DISEASE

SARS-CoV-2, the causative agent of COVID-19, is currently generating a global pandemic. So far, dexamethasone and remdesivir have shown efficacy in adequately powered clinical trials. In addition, passive immunotherapy appears as a promising therapeutic approach, particularly for early stages of the disease in which patients have not yet established their specific immune response. Different anti-receptor binding domain (RBD) human monoclonal antibodies (mAbs) have been evaluated in the treatment of COVID-19. It has been previously shown that the RBD from the viral spike glycoprotein elicits high titers of NAbs against SARS-CoV-2 when used as immunogen in horses. In this regard, equine polyclonal antibodies (EpAbs) can represent a practical and efficient source of NAbs. EpAbs are composed of F(ab)'2 fragments generated by pepsin digestion. These fragments retain the bivalent binding capacity of IgG immunoglobulins but lack the constant region (Fc), responsible for serum sickness reactions and Fc-triggered side effects. EpAbs recognize a vast array of epitopes (limiting the risk of viral escape mutations) and tend to develop greater avidity than mAbs for their cognate antigens. In addition, EpAbs are relatively easy to manufacture allowing a fast development and scaling up for a treatment. We have previously described the development and in vitro characterization of a therapeutic based on purified equine anti-RBD F(ab)2 fragments, called INM005. INM005 shows a very high serum neutralization titer against SARS-CoV-2. We conducted a phase 2/3 clinical to test the therapeutic effect of INM005 on COVID-19 patients. Albeit not having reached the primary endpoint, we found clinical improvement of hospitalized patients with SARS-CoV-2 pneumonia, particularly those with severe disease. Rate of improvement in at least two categories was statistically significantly higher for INM005 at days 14 and 21 of follow-up. Time to improvement in two ordinal categories or hospital discharge was 14·2 (± 0·7) days in the INM005 group and 16·3 (± 0·7) days in the placebo group. Subgroup analyses showed a beneficial effect of INM005 over severe patients and in those with negative baseline antibodies. Overall mortality was 6·9% the INM005 group and 11·4% in the placebo group. Adverse events of special interest were mild or moderate;no anaphylaxis was reported. Based on these results, ANMAT granted the emergency use approval of INM005 to treat hospitalized COVID-19 severe patients. Following approval, more than 20,000 patients have been treated with INM005. We will be presenting the results of the "real world use of this immunotherapy during the second wave of the pandemics in Argentina.

The dynamic nature of the coronavirus receptor, angiotensin-converting enzyme 2 (ACE2) in differentiating airway epithelia.

Once inhaled, SARS-CoV-2 particles enter respiratory ciliated cells by interacting with angiotensin converting enzyme 2 (ACE2). Understanding the nature of ACE2 within airway tissue has become a recent focus particularly in light of the COVID-19 pandemic. Airway mucociliary tissue was generated in-vitro using primary human nasal epithelial cells and the air-liquid interface (ALI) model of differentiation. Using ALI tissue, three distinct transcript variants of ACE2 were identified. One transcript encodes the documented full-length ACE2 protein. The other two transcripts are unique truncated isoforms, that until recently had only been predicted to exist via sequence analysis software. Quantitative PCR revealed that all three transcript variants are expressed throughout differentiation of airway mucociliary epithelia. Immunofluorescence analysis of individual ACE2 protein isoforms exogenously expressed in cell-lines revealed similar abilities to localize in the plasma membrane and interact with the SARS CoV 2 spike receptor binding domain. Immunohistochemistry on differentiated ALI tissue using antibodies to either the N-term or C-term of ACE2 revealed both overlapping and distinct signals in cells, most notably only the ACE2 C-term antibody displayed plasma-membrane localization. We also demonstrate that ACE2 protein shedding is different in ALI Tissue compared to ACE2-transfected cell lines, and that ACE2 is released from both the apical and basal surfaces of ALI tissue. Together, our data highlights various facets of ACE2 transcripts and protein in airway mucociliary tissue that may represent variables which impact an individual's susceptibility to SARS-CoV-2 infection, or the severity of Covid-19.

XAV-19, a Swine Glyco-Humanized Polyclonal Antibody Against SARS-CoV-2 Spike Receptor-Binding Domain, Targets Multiple Epitopes and Broadly Neutralizes Variants.

Amino acid substitutions and deletions in the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can reduce the effectiveness of monoclonal antibodies (mAbs). In contrast, heterologous polyclonal antibodies raised against S protein, through the recognition of multiple target epitopes, have the potential to maintain neutralization capacities. XAV-19 is a swine glyco-humanized polyclonal neutralizing antibody raised against the receptor binding domain (RBD) of the Wuhan-Hu-1 Spike protein of SARS-CoV-2. XAV-19 target epitopes were found distributed all over the RBD and particularly cover the receptor binding motives (RBMs), in direct contact sites with the angiotensin converting enzyme-2 (ACE-2). Therefore, in Spike/ACE-2 interaction assays, XAV-19 showed potent neutralization capacities of the original Wuhan Spike and of the United Kingdom (Alpha/B.1.1.7) and South African (Beta/B.1.351) variants. These results were confirmed by cytopathogenic assays using Vero E6 and live virus variants including the Brazil (Gamma/P.1) and the Indian (Delta/B.1.617.2) variants. In a selective pressure study on Vero E6 cells conducted over 1 month, no mutation was associated with the addition of increasing doses of XAV-19. The potential to reduce viral load in lungs was confirmed in a human ACE-2 transduced mouse model. XAV-19 is currently evaluated in patients hospitalized for COVID-19-induced moderate pneumonia in phase 2a-2b (NCT04453384) where safety was already demonstrated and in an ongoing 2/3 trial (NCT04928430) to evaluate the efficacy and safety of XAV-19 in patients with moderate-to-severe COVID-19. Owing to its polyclonal nature and its glyco-humanization, XAV-19 may provide a novel safe and effective therapeutic tool to mitigate the severity of coronavirus disease 2019 (COVID-19) including the different variants of concern identified so far.

Generation and Effect Testing of a SARS-CoV-2 RBD-Targeted Polyclonal Therapeutic Antibody Based on a 2-D Airway Organoid Screening System.

Coronavirus disease 2019 (COVID-19) is an acute respiratory infectious disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The US FDA has approved several therapeutics and vaccines worldwide through the emergency use authorization in response to the rapid spread of COVID-19. Nevertheless, the efficacies of these treatments are being challenged by viral escape mutations. There is an urgent need to develop effective treatments protecting against SARS-CoV-2 infection and to establish a stable effect-screening model to test potential drugs. Polyclonal antibodies (pAbs) have an intrinsic advantage in such developments because they can target rapidly mutating viral strains as a result of the complexity of their binding epitopes. In this study, we generated anti-receptor-binding domain (anti-RBD) pAbs from rabbit serum and tested their safety and efficacy in response to SARS-CoV-2 infection both in vivo and ex vivo. Primary human bronchial epithelial two-dimensional (2-D) organoids were cultured and differentiated to a mature morphology and subsequently employed for SARS-CoV-2 infection and drug screening. The pAbs protected the airway organoids from viral infection and tissue damage. Potential side effects were tested in mouse models for both inhalation and vein injection. The pAbs displayed effective viral neutralization effects without significant side effects. Thus, the use of animal immune serum-derived pAbs might be a potential therapy for protection against SARS-CoV-2 infection, with the strategy developed to produce these pAbs providing new insight into the treatment of respiratory tract infections, especially for infections with viruses undergoing rapid mutation.