Virus Neutralization Research Articles

In Silico Design and Evaluation of a Novel Therapeutic Agent Against the Spike Protein as a Novel Treatment Strategy for COVID-19 Treatment.

Coronavirus disease 2019 (COVID-19) is a viral respiratory disease that is associated with severe damage to other human organs. It causes by a novel coronavirus, and it is spreading all over the world. To date, there is some approved vaccine or therapeutic agent which could be effective against this disease. But their effectiveness against mutated strains is not studied completely. The spike glycoprotein on the surface of the coronaviruses gives the virus the ability to bind to host cell receptors and enter cells. Inhibition of attachment of these spikes can lead to virus neutralization by inhibiting viral entrance. In this study, we tried to use the virus entrance strategy against itself by utilizing virus receptor (ACE-2) in order to design an engineered protein consisting of a human Fc antibody fragment and a part of ACE-2, which reacts with virus RBD, and we also evaluated this interaction by computational methods and in silico methods. Subsequently, we have designed a new protein structure to bind with this site and inhibit the virus from attaching to its cell receptor, mechanically or chemically. Various in silico software, bioinformatics, and patent databases were used to retrieve the requested gene and protein sequences. The physicochemical properties and possibility of allergenicity were also examined. Three-dimensional structure prediction and molecular docking were also performed to develop the most suitable therapeutic protein. The designed protein consisted of a total of 256 amino acids with a molecular weight of 28984.62 and 5.92 as a theoretical isoelectric point. Instability and aliphatic index and grand average of hydropathicity are 49.99, 69.57 and -0.594, respectively. In silico studies can provide a good opportunity to study viral proteins and new drugs or compounds since they do not need direct exposure to infectious agents or equipped laboratories. The suggested therapeutic agent should be further characterized in vitro and in vivo.

Optimization and validation of a virus-like particle pseudotyped virus neutralization assay for SARS-CoV-2.

Spike-protein-based pseudotyped viruses were used to evaluate vaccines during the COVID-19 pandemic. However, they cannot be used to evaluate the envelope (E), membrane (M), and nucleocapsid (N) proteins. The first generation of virus-like particle (VLP) pseudotyped viruses contains these four structural proteins, but their titers for wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are relatively low, even lower for the omicron variant, rendering them unsuitable for neutralizing antibody detection. By optimizing the spike glycoprotein signal peptide, substituting the complexed M and E proteins with SARS-COV-1, optimizing the N protein with specific mutations (P199L, S202R, and R203M), and truncating the packaging signal, PS9, we increased the titer of the wild-type VLP pseudotyped virus over 100-fold, and successfully packaged the omicron VLP pseudotyped virus. The SARS-CoV-2 VLP pseudotyped viruses maintained stable titers, even through 10 freeze-thaw cycles. The key neutralization assay parameters were optimized, including cell type, cell number, and viral inoculum. The assay demonstrated minimal variation in both intra- and interassay results, at 11.5% and 11.1%, respectively. The correlation between the VLP pseudotyped virus and the authentic virus was strong (r=0.9). Suitable for high-throughput detection of various mutant strains in clinical serum. In summary, we have developed a reliable neutralization assay for SARS-CoV-2 based on VLP pseudotyped virus.

Randomized, blind, controlled phase III clinical trial: Assessing the immunogenicity and safety of freeze-dried human rabies vaccine (vero cell) with a 4-dose regimen (2-1-1) in a 10–60 year-old demographic

ObjectiveThe aim of this study is to demonstrate that the freeze-dried human rabies vaccine (Vero cell), administered in a four-dose schedule (2-1-1) to the 10–60 years old population, has immunogenicity that is not inferior to the approved five-dose schedule and similar vaccines with a four-dose schedule, and to evaluate its safety. MethodA total of 1800 individuals were enrolled and divided into three groups: four-dose test group, four-dose control group, and five-dose control group. The rabies virus neutralizing antibodies were measured using the Rapid Fluorescent Focus Inhibition Test to assess immunogenicity, and the incidence of adverse events and serious adverse events were statistically analyzed. ResultsThe seroconversion rates 14 days after the first dose and 14 days after the complete course of vaccination were 100% in all three groups. The antibody GMC of the four-dose test group was higher than that of the five-dose control group, but slightly lower than the four-dose control group. Seven days after the first dose, both four-dose regimen groups showed higher seroconversion rates and antibody GMCs compared to the five-dose regimen group, proving that the immunogenic effect of the four-dose regimen seven days post-first vaccination is superior to the five-dose regimen. The overall incidence of adverse events showed no significant difference between the four-dose test group and the five-dose control group, but was significantly lower in the four-dose test group compared to the four-dose control group. ConclusionThe vaccine in the four-dose test group is equivalent in immunogenic effect to the four-dose control group vaccine and superior to the five-dose control group vaccine; the safety of the vaccine in the four-dose test group is equivalent to the five-dose control group vaccine and superior to the four-dose control group vaccine. clinicaltrials.gov number: NCT05549908.

Development of a highly effective combination monoclonal antibody therapy against Herpes simplex virus

BackgroundInfections with Herpes simplex virus (HSV)-1 or -2 usually present as mild chronic recurrent disease, however in rare cases can result in life-threatening conditions with a large spectrum of pathology. Monoclonal antibody therapy has great potential especially to treat infections with virus resistant to standard therapies. HDIT101, a humanized IgG targeting HSV-1/2 gB was previously investigated in phase 2 clinical trials. The aim of this study was to develop a next-generation therapy by combining different antiviral monoclonal antibodies.MethodsA lymph-node derived phage display library (LYNDAL) was screened against recombinant gB from Herpes simplex virus (HSV) -1 and HDIT102 scFv was selected for its binding characteristics using bio-layer interferometry. HDIT102 was further developed as fully human IgG and tested alone or in combination with HDIT101, a clinically tested humanized anti-HSV IgG, in vitro and in vivo. T-cell stimulating activities by antigen-presenting cells treated with IgG-HSV immune complexes were analyzed using primary human cells. To determine the epitopes, the cryo-EM structures of HDIT101 or HDIT102 Fab bound to HSV-1F as well as HSV-2G gB protein were solved at resolutions < 3.5 Å.ResultsHDIT102 Fab showed strong binding to HSV-1F gB with Kd of 8.95 × 10–11 M and to HSV-2G gB with Kd of 3.29 × 10–11 M. Neutralization of cell-free virus and inhibition of cell-to-cell spread were comparable between HDIT101 and HDIT102. Both antibodies induced internalization of gB from the cell surface into acidic endosomes by binding distinct epitopes in domain I of gB and compete for binding. CryoEM analyses revealed the ability to form heterogenic immune complexes consisting of two HDIT102 and one HDIT101 Fab bound to one gB trimeric molecule. Both antibodies mediated antibody-dependent phagocytosis by antigen presenting cells which stimulated autologous T-cell activation. In vivo, the combination of HDIT101 and HDIT102 demonstrated synergistic effects on survival and clinical outcome in immunocompetent BALB/cOlaHsd mice.ConclusionThis biochemical and immunological study showcases the potential of an effective combination therapy with two monoclonal anti-gB IgGs for the treatment of HSV-1/2 induced disease conditions.

Alpha and gamma mangostins inhibit wild-type B SARS-CoV-2 more effectively than the SARS-CoV-2 variants and the major target is unlikely the 3C-like protease

BackgroundAnti-SARS-CoV-2 and immunomodulatory drugs are important for treating clinically severe patients with respiratory distress symptoms. Alpha- and gamma-mangostins (AM and GM) were previously reported as potential 3C-like protease (3CLpro) and Angiotensin-converting enzyme receptor 2 (ACE2)-binding inhibitors in silico. ObjectiveWe aimed to evaluate two active compounds, AM and GM, from Garcinia mangostana for their antivirals against SARS-CoV-2 in live virus culture systems and their cytotoxicities using standard methods. Also, we aimed to prove whether 3CLpro and ACE2 neutralization were major targets and explored whether any additional targets existed. MethodsWe tested the translation and replication efficiencies of SARS-CoV-2 in the presence of AM and GM. Initial and subgenomic translations were evaluated by immunofluorescence of SARS-CoV-2 3CLpro and N expressions at 16 h after infection. The viral genome was quantified and compared with the untreated group. We also evaluated the efficacies and cytotoxicities of AM and GM against four strains of SARS-CoV-2 (wild-type B, B.1.167.2, B.1.36.16, and B.1.1.529) in Vero E6 cells. The potential targets were evaluated using cell-based anti-attachment, time-of-drug addition, in vitro 3CLpro activities, and ACE2-binding using a surrogated viral neutralization test (sVNT). Moreover, additional targets were explored using combinatorial network-based interactions and Chemical Similarity Ensemble Approach (SEA). ResultsAM and GM reduced SARS-CoV-2 3CLpro and N expressions, suggesting that initial and subgenomic translations were globally inhibited. AM and GM inhibited all strains of SARS-CoV-2 at EC50 of 0.70–3.05 μM, in which wild-type B was the most susceptible strain (EC50 0.70–0.79 μM). AM was slightly more efficient in the variants (EC50 0.88–2.41 μM), resulting in higher selectivity indices (SI 3.65–10.05), compared to the GM (EC50 0.94–3.05 μM, SI 1.66–5.40). GM appeared to be more toxic than AM in both Vero E6 and Calu-3 cells. Cell-based anti-attachment and time-of-addition suggested that the potential molecular target could be at the post-infection. 3CLpro activity and ACE2 binding were interfered with in a dose-dependent manner but were insufficient to be a major target. Combinatorial network-based interaction and chemical similarity ensemble approach (SEA) suggested that fatty acid synthase (FASN), which was critical for SARS-CoV-2 replication, could be a target of AM and GM. ConclusionAM and GM inhibited SARS-CoV-2 with the highest potency at the wild-type B and the lowest at the B.1.1.529. Multiple targets were expected to integratively inhibit viral replication in cell-based system.

Multiparametric analysis of the specific immune response against SARS-CoV-2

Background SARS-CoV-2, which causes COVID-19, has killed more than 7 million people worldwide. Understanding the development of postinfectious and postvaccination immune responses is necessary for effective treatment and the introduction of appropriate antipandemic measures. Objectives We analysed humoral and cell-mediated anti-SARS-CoV-2 immune responses to spike (S), nucleocapsid (N), membrane (M), and open reading frame (O) proteins in individuals collected up to 1.5 years after COVID-19 onset and evaluated immune memory. Methods Peripheral blood mononuclear cells and serum were collected from patients after COVID-19. Sampling was performed in two rounds: 3-6 months after infection and after another year. Most of the patients were vaccinated between samplings. SARS-CoV-2-seronegative donors served as controls. ELISpot assays were used to detect SARS-CoV-2-specific T and B cells using peptide pools (S, NMO) or recombinant proteins (rS, rN), respectively. A CEF peptide pool consisting of selected viral epitopes was applied to assess the antiviral T-cell response. SARS-CoV-2-specific antibodies were detected via ELISA and a surrogate virus neutralisation assay. Results We confirmed that SARS-CoV-2 infection induces the establishment of long-term memory IgG+ B cells and memory T cells. We also found that vaccination enhanced the levels of anti-S memory B and T cells. Multivariate comparison also revealed the benefit of repeated vaccination. Interestingly, the T-cell response to CEF was lower in patients than in controls. Conclusion This study supports the importance of repeated vaccination for enhancing immunity and suggests a possible long-term perturbation of the overall antiviral immune response caused by SARS-CoV-2 infection.

Exposure of cattle to tick-borne encephalitis virus in the historical endemic zone in north-eastern France

BackgroundTick-borne encephalitis (TBE) is a severe human neuroinfection caused by TBE virus (TBEV). TBEV is transmitted by tick bites and by the consumption of unpasteurized dairy products from infected asymptomatic ruminants. In France, several food-borne transmission events have been reported since 2020, raising the question of the level of exposure of domestic ungulates to TBEV. In this study, our objectives were (i) to estimate TBEV seroprevalence and quantify antibodies titres in cattle in the historical endemic area of TBEV in France using the micro virus neutralisation test (MNT) and (ii) to compare the performance of two veterinary cELISA kits with MNT for detecting anti-TBEV antibodies in cattle in various epidemiological contexts. A total of 344 cattle sera from four grid cells of 100 km² in Alsace-Lorraine (endemic region) and 84 from western France, assumed to be TBEV-free, were investigated.ResultsIn Alsace-Lorraine, cattle were exposed to the virus with an overall estimated seroprevalence of 57.6% (95% CI: 52.1–62.8%, n = 344), varying locally from 29.9% (95% CI: 21.0–40.0%) to 92.1% (95% CI: 84.5–96.8%). Seroprevalence did not increase with age, with one- to three-year-old cattle being as highly exposed as older ones, suggesting a short-life duration of antibodies. The proportion of sera with MNT titres lower than 1:40 per grid cell decreased with increased seroprevalence. Both cELISA kits showed high specificity (> 90%) and low sensitivity (less than 78.1%) compared with MNT. Sensitivity was lower for sera with neutralising antibodies titres below 1:40, suggesting that sensitivity of these tests varied with local virus circulation intensity.ConclusionsOur results highlight that cattle were highly exposed to TBEV. Screening strategy and serological tests should be carefully chosen according to the purpose of the serological study and with regard to the limitations of each method.

Highly sensitive and quantitative HiBiT-tagged Nipah virus-like particles: A platform for rapid antibody neutralization studies

Biocontainment regulations restrict the research on NiV to BSL-4 laboratories, thus limiting the mechanistic studies related to viral entry and allied pathogenesis. Understanding the precise process of viral-particle production and host cell entry is critical for designing targeted therapies or particle-based vaccines. In this study, we have synthesized HiBiT-tagged-NiV-VLPs to ease in-vitro BSL-2 particle handling. We propose a simple yet effective approach of generating substantial amount of HiBiT-tagged NiV-VLPs in vitro by co-expressing viral structural proteins in HEK293T cells. Though homologous to parent virus, the incapacitated replication potential facilitates a BSL-2 handling of these particles. The inclusion of a highly sensitive HiBiT tag on these VLPs allows for a quick detection of viral binding and entry, as well as in assessing the efficiency of neutralizing antibodies in vitro using the NanoBiT technology. The HiBiT-tag binds in high affinity with LgBiT (Large BiT an 18 kDa fusion protein and complementary subunit of HiBiT peptide), and the resultant complex elicits high intensity luminescence in the presence of substrate. The VLPs produced were morphologically and functionally identical to the native virus, and the HiBiT-tag permitted their quick application in viral binding, entry, and antibody neutralization assays.“Thus, we report a simple setting for generating HiBiT-NiV VLPs which can be utilized in a BSL-2 laboratory, to concurrently quantify features of NiV assembly, binding and entry. This also offers an alternate-safe and effective platform for viral based antibody neutralization assays in vitro”.

Evaluation of longitudinal passive immunity transfer against lumpy skin disease virus in calves by different serological methods

To implement effective lumpy skin disease (LSD) control measures, such as timely vaccination, particularly in calves and serological monitoring, it is necessary to evaluate immune response after vaccination, both in adult cattle and in their calves. The aim of this study was to evaluate passive immunity transfer and duration of maternal antibodies against lumpy skin disease virus (LSDV) in calves born to vaccinated cows by two different serological methods. The longitudinal study was carried out on two farms in Serbia where no cases were reported during LSD outbreak in 2016. Fifteen cows on each farm were vaccinated and revaccinated with attenuated vaccine - Neethling strain. A total of 30 cows and 30 calves on both farms were included in the study. Serum samples from cows were collected on calving day and serum samples from their respective calves on days 10, 20, 30, 45, 60, 75, 90, 105 and 120 after birth. Colostrum samples were collected only from 15 cows on one farm. In order to determine the presence of antibodies against LSDV a total of 30 cow sera samples, 15 colostrum samples and 270 calf sera samples were examined by commercial enzyme-linked immunosorbent assay (ELISA) and modified virus neutralization test (VNT). Overall, the performance of both serological tests was very satisfactory. The results of this longitudinal study showed that persistence of passive immunity in calves is less than 4 months, and that most calves are not protected against LSDV at that age. Since the vaccination is the most important control measure against LSDV, the recommended age of six months for vaccination of calves born to vaccinated cows should be reassessed to achieve the most optimal protection against LSD.

Persistence of passive immunity in calves receiving colostrum from cows vaccinated with a live attenuated lumpy skin disease vaccine and the performance of serological tests.

This study aimed to determine the persistent duration of maternal immunity against lumpy skin disease virus (LSDV) in dairy calves born from vaccinated cows using a virus neutralization test (VNT). The performance of the VNT and an in-house-ELISA test was also determined. Thirty-seven pregnant cows from 12 LSD-free dairy farms in Lamphun province, Thailand were immunized with a homologous Neethling strain-based attenuated vaccine and calved from December 2021 to April 2022. Blood samples from dam-calve pairs were collected within the first week after calving. Subsequently, blood samples were taken from the calves at monthly intervals over a period of 4 months and tested for the humoral immune response using a VNT. The calf sera were also tested with an in-house ELISA test to estimate the accuracy of both tests using a Bayesian approach. For the results, antibodies against LSDV can persist in cows for 4-9 months post-vaccination. Moreover, neutralizing antibodies and LSDV-specific antibodies against LSDV were detected in the majority of calves (75.68%) during the first week after colostrum intake. However, the percentage of seropositive calves declined to zero by day 120, with seropositivity dropping below 50% after day 60. Only a small number of seropositive calves (approximately 13.51%) were observed on day 90. These findings indicated that passive immunity against LSDV can last up to 3 months. The median of posterior estimates for sensitivity (Se) and specificity (Sp) of the VNT were 87.3% [95% posterior probability interval (PPI) = 81.1-92.2%] and 94.5% (95% PPI = 87.7-98.3%), respectively. The estimated Se and Sp for the ELISA test were 83.1% (95% PPI = 73.6-92.6%) and 94.7% (95% PPI = 88.4-98.5%), respectively. In conclusion, this study illustrates the transfer and persistence of maternal passive immunity against LSDV to calves under field conditions. This highlights a potential three-month vaccination gap in calves born from vaccinated cows, while an in-house ELISA test can be used as an ancillary test for LSDV immune response detection. However, further research is required to assess the vaccination protocols for calves as young as 2 months old to precisely determine the duration of maternal immunity.

Does Perioperative Administration of Rabies Vaccine in Dogs Undergoing Surgical Sterilization Induce an Adequate Antibody Response?

High-volume spay/neuter events may facilitate access to free-roaming dogs to admi-nister rabies vaccination, but important questions remain regarding the effect of surgery and anesthesia on the immune response to a vaccine administered in the perioperative period. This study evaluated the immunogenicity of primary rabies vaccination in dogs when administered during the immediate perioperative period at the time of surgical sterilization (ovariohysterectomy/orchidectomy). Healthy dogs of both sexes presenting for surgical sterilization who had never been vaccinated against rabies virus were eligible for enrollment in the study. Fifty dogs ranging in age from 5 to 96 months were enrolled and were vaccinated against rabies virus during the recovery period following anesthesia and surgery. Rabies virus neutralizing antibody (RVNA) titers were measured preoperatively and 28 days postoperatively. This cohort was compared to a historical control cohort of 57 dogs who received primary rabies vaccination for travel purposes and had RVNA titers measured at the same laboratory as the study group 28-35 days post-vaccination. After controlling for age and sex, there was no statistically significant difference in immunogenicity of a rabies vaccine administered to dogs during the perioperative period in comparison to dogs that received the rabies vaccine for travel alone in the absence of surgery. Perioperative administration of a rabies vaccine in dogs under-going surgical sterilization induces an adequate antibody response. We recommend that rabies vaccine be administered perioperatively during spay/neuter campaigns in canine rabies endemic areas if other opportunities to ac-cess veterinary care and rabies vaccination are limited.

Accuracy of Anti-SARS-CoV-2 Antibody in Comparison with Surrogate Viral Neutralization Test in Persons Living with HIV, Systemic Lupus Erythematosus, and Chronic Kidney Disease.

The presence of the anti-SARS-CoV-2-RBD antibody (anti-RBD) prevents severe COVID-19. We aimed to determine the accuracy of a point-of-care anti-RBD testing implemented in persons living with HIV (PLWH), systemic lupus erythematosus (SLE), and chronic kidney disease (CKD). We enrolled 182 non-comorbid subjects and 335 comorbid subjects (PLWH, SLE, CKD) to test the anti-RBD assay compared to the surrogate viral neutralization test (sVNT) as the reference test. We performed linear correlation analysis between anti-RBD and sVNT, along with an ROC analysis to ascertain the anti-RBD cutoff at 30%, 60%, and 90% inhibition of sVNT, to calculate accuracy. The correlations between anti-RBD and sVNT among all groups were excellent, with R = 0.7903, R = 0.7843, and R = 0.8153 among the non-comorbid, SLE, and CKD groups, respectively, and with significantly higher correlation among the PLWH group (R = 0.8877; p-value = 0.0072) compared to the non-comorbid group. The accuracy of the anti-RBD test among the PLWH and CKD groups was similar to that among the non-comorbid group but showed lower sensitivity in the SLE group (p = 0.000014). The specificity of the test remained high in all groups. In conclusion, the anti-RBD test had excellent correlation with the sVNT. The persistently high specificity in all groups suggests that this test can be reliably utilized to detect the presence of low neutralization capacity, prompting additional vaccination.

SARS-CoV-2 Neutralization Assays Used in Clinical Trials: A Narrative Review.

Since the emergence of COVID-19, extensive research efforts have been undertaken to accelerate the development of multiple types of vaccines to combat the pandemic. These include inactivated, recombinant subunit, viral vector, and nucleic acid vaccines. In the development of these diverse vaccines, appropriate methods to assess vaccine immunogenicity are essential in both preclinical and clinical studies. Among the biomarkers used in vaccine evaluation, the neutralizing antibody level serves as a pivotal indicator for assessing vaccine efficacy. Neutralizing antibody detection methods can mainly be classified into three types: the conventional virus neutralization test, pseudovirus neutralization test, and surrogate virus neutralization test. Importantly, standardization of these assays is critical for their application to yield results that are comparable across different laboratories. The development and use of international or regional standards would facilitate assay standardization and facilitate comparisons of the immune responses induced by different vaccines. In this comprehensive review, we discuss the principles, advantages, limitations, and application of different SARS-CoV-2 neutralization assays in vaccine clinical trials. This will provide guidance for the development and evaluation of COVID-19 vaccines.

Direct comparison of SARS-CoV-2 variant specific neutralizing antibodies in human and hamster sera

Antigenic characterization of newly emerging SARS-CoV-2 variants is important to assess their immune escape and judge the need for future vaccine updates. To bridge data obtained from animal sera with human sera, we analyzed neutralizing antibody titers in human and hamster single infection sera in a highly controlled setting using the same authentic virus neutralization assay performed in one laboratory. Using a Bayesian framework, we found that titer fold changes in hamster sera corresponded well to human sera and that hamster sera generally exhibited higher reactivity.

Hepatitis C Virus E1E2 Structure, Diversity, and Implications for Vaccine Development.

Hepatitis C virus (HCV) is a major medical health burden and the leading cause of chronic liver disease and cancer worldwide. More than 58 million people are chronically infected with HCV, with 1.5 million new infections occurring each year. An effective HCV vaccine is a major public health and medical need as recognized by the World Health Organization. However, due to the high variability of the virus and its ability to escape the immune response, HCV rapidly accumulates mutations, making vaccine development a formidable challenge. An effective vaccine must elicit broadly neutralizing antibodies (bnAbs) in a consistent fashion. After decades of studies from basic research through clinical development, the antigen of choice is considered the E1E2 envelope glycoprotein due to conserved, broadly neutralizing antigenic domains located in the constituent subunits of E1, E2, and the E1E2 heterodimeric complex itself. The challenge has been elicitation of robust humoral and cellular responses leading to broad virus neutralization due to the relatively low immunogenicity of this antigen. In view of this challenge, structure-based vaccine design approaches to stabilize key antigenic domains have been hampered due to the lack of E1E2 atomic-level resolution structures to guide them. Another challenge has been the development of a delivery platform in which a multivalent form of the antigen can be presented in order to elicit a more robust anti-HCV immune response. Recent nanoparticle vaccines are gaining prominence in the field due to their ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both the cellular and humoral components of the immune system. This review focuses on recent advances in understanding the E1E2 heterodimeric structure to facilitate a rational design approach and the potential for development of a multivalent nanoparticle-based HCV E1E2 vaccine. Both aspects are considered important in the development of an effective HCV vaccine that can effectively address viral diversity and escape.

Safety and immunogenicity of COReNAPCIN, a SARS-CoV-2 mRNA vaccine, as a fourth heterologous booster in healthy Iranian adults: A double-blind, randomized, placebo-controlled, phase 1 clinical trial with a six-month follow-up

The recurrent COVID-19 infection, despite global vaccination, highlights the need for booster doses. A heterologous booster has been suggested to enhance immunity and protection against emerging variants of concern of the SARS-CoV-2 virus. In this report, we aimed to assess the safety, and immunogenicity of COReNAPCIN, as a fourth booster dose after three doses of inactivated vaccines. MethodsThe study was conducted as a double-blind, randomized, placebo-controlled phase 1 clinical trial of the mRNA-based vaccine candidate, COReNAPCIN. The vaccine was injected as a heterologous booster in healthy Iranian adults aged 18–50 who had previously received three doses of inactivated SARS-CoV-2 vaccines. In the study, 30 participants were randomly assigned to receive either COReNAPCIN in two different doses (25 µg and 50 µg) or placebo. The vaccine candidate contained mRNA encoding the complete sequence of the pre-fusion stabilized Spike protein of SARS-CoV-2, formulated within lipid nanoparticles. The primary endpoint was safety and the secondary objective was humoral immunogenicity until 6 months post-vaccination. The cellular immunogenicity was pursued as an exploratory outcome. ResultsCOReNAPCIN was well tolerated in vaccinated individuals in both doses with no life-threatening or other serious adverse events. The most noticeable solicited adverse events were pain at the site of injection, fatigue and myalgia. Regarding the immunogenicity, despite the seroprevalence of SARS-CoV-2 antibodies due to the vaccination history for all and previous SARS-CoV-2 infection for some participants, the recipients of 25 and 50 µg COReNAPCIN, two weeks post-vaccination, showed 6·6 and 8·1 fold increase in the level of anti-RBD, and 11·5 and 21·7 fold increase in the level of anti-spike antibody, respectively. The geometric mean virus neutralizing titers reached 10.2 fold in the 25 µg group and 8.4 fold in 50 µg group of pre-boost levels. After 6 months, the measured anti-spike antibody concentration still maintains a geometric mean fold rise of 2.8 and 6.3, comparing the baseline levels in 25 and 50 µg groups, respectively. Additionally, the significant increase in the spike-specific IFN-ϒ T-cell response upon vaccination underscores the activation of cellular immunity. ConclusionCOReNAPCIN booster showed favorable safety, tolerability, and immunogenicity profile, supporting its further clinical development (Trial registration: IRCT20230131057293N1).

Antibody responses in blood and saliva post COVID-19 bivalent booster do not reveal an Omicron BA.4/BA.5- specific response.

Current SARS-CoV-2 strains continue to mutate and attempt to evade the antibody response elicited by previous exposures and vaccinations. In September of 2022, the first updated SARS-CoV-2 vaccines, designed to create immune responses specific for the variants circulating in 2022, were approved. These new vaccines, known commonly as the bivalent boost(er), include mRNA that encodes both the original Wuhan-Hu-1 spike protein as well as the spike protein specific to the Omicron BA.4 and BA.5 variants. We recruited volunteers from University of Massachusetts student, faculty and staff members to provide samples of blood and saliva at four different time points, including pre-boost and three times post boost and analyzed samples for antibody production as well as neutralization of virus. Our data provide a comprehensive analysis of the antibody response following a single dose of the bivalent boost over a 6-month period and support previous findings that the response induced after the bivalent boost does not create a strong BA.4/BA.5-specific antibody response. We found no evidence of a specific anti-BA.4/BA.5 response developing over time, including in a sub-population of individuals who become infected after a single dose of the bivalent booster. Additionally, we present data that support the use of saliva samples as a reliable alternative to blood for antibody detection against specific SARS-CoV-2 antigens.

Comparison of Five Serological Methods for the Detection of West Nile Virus Antibodies

The West Nile Virus (WNV), a member of the family Flaviviridae, is an emerging mosquito-borne flavivirus causing potentially severe infections in humans and animals involving the central nervous system (CNS). Due to its emerging tendency, WNV now occurs in many areas where other flaviviruses are co-occurring. Cross-reactive antibodies with flavivirus infections or vaccination (e.g., tick-borne encephalitis virus (TBEV), Usutu virus (USUV), yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV)) therefore remain a major challenge in diagnosing flavivirus infections. Virus neutralization tests are considered as reference tests for the detection of specific flavivirus antibodies, but are elaborate, time-consuming and need biosafety level 3 facilities. A simple and straightforward assay for the differentiation and detection of specific WNV IgG antibodies for the routine laboratory is urgently needed. In this study, we compared two commercially available enzyme-linked immunosorbent assays (anti-IgG WNV ELISA and anti-NS1-IgG WNV), a commercially available indirect immunofluorescence assay, and a newly developed in-house ELISA for the detection of WNV-NS1-IgG antibodies. All four tests were compared to an in-house NT to determine both the sensitivity and specificity of the four test systems. None of the assays could match the specificity of the NT, although the two NS1-IgG based ELISAs were very close to the specificity of the NT at 97.3% and 94.6%. The in-house WNV-NS1-IgG ELISA had the best performance regarding sensitivity and specificity. The specificities of the ELISA assays and the indirect immunofluorescence assays could not meet the necessary specificity and/or sensitivity.

Efficacy and safety of novel multifunctional M10 CAR-T cells in HIV-1-infected patients: a phase I, multicenter, single-arm, open-label study

Chimeric antigen receptor T (CAR-T) cells have been proposed for HIV-1 treatment but have not yet demonstrated desirable therapeutic efficacy. Here, we report newly developed anti-HIV-1 CAR-T cells armed with endogenic broadly neutralizing antibodies (bNAbs) and the follicle-homing receptor CXCR5, termed M10 cells. M10 cells were designed to exercise three-fold biological functions, including broad cytotoxic effects on HIV-infected cells, neutralization of cell-free viruses produced after latency reversal, and B-cell follicle homing. After demonstrating the three-fold biological activities, M10 cells were administered to treat 18 HIV-1 patients via a regimen of two allogenic M10 cell infusions with an interval of 30 days, with each M10 cell infusion followed by two chidamide stimulations for HIV-1 reservoir activation. Consequently, 74.3% of M10 cell infusions resulted in significant suppression of viral rebound, with viral loads declining by an average of 67.1%, and 10 patients showed persistently reduced cell-associated HIV-1 RNA levels (average decrease of 1.15 log10) over the 150-day observation period. M10 cells were also found to impose selective pressure on the latent viral reservoir. No significant treatment-related adverse effects were observed. Overall, our study supported the potential of M10 CAR-T cells as a novel, safe, and effective therapeutic option for the functional cure of HIV-1/AIDS.

Equine herpesvirus type 1 (EHV-1) replication at the upper respiratory entry site is inhibited by neutralizing EHV-1-specific IgG1 and IgG4/7 mucosal antibodies.

Equine herpesvirus type 1 (EHV-1) is a contagious respiratory pathogen that infects the mucosa of the upper respiratory tract (URT). Mucosal immune responses at the URT provide the first line of defense against EHV-1 and are crucial for orchestrating immunity. To define host-pathogen interactions, we characterized B-cell responses, antibody isotype functions, and EHV-1 replication of susceptible (non-immune) and clinically protected (immune) horses after experimental EHV-1 infection. Nasal secretion and nasal wash samples were collected and used for the isolation of DNA, RNA, and mucosal antibodies. Shedding of infectious virus, EHV-1 copy numbers, viral RNA expression, and host B-cell activation in the URT were compared based on host immune status. Mucosal EHV-1-specific antibody responses were associated with EHV-1 shedding and viral RNA transcription. Finally, mucosal immunoglobulin G (IgG) and IgA isotypes were purified and tested for neutralizing capabilities. IgG1 and IgG4/7 neutralized EHV-1, while IgG3/5, IgG6, and IgA did not. Immune horses secreted high amounts of mucosal EHV-1-specific IgG4/7 antibodies and quickly upregulated B-cell pathway genes, while EHV-1 was undetected by virus isolation and PCR. RNA transcription analysis reinforced incomplete viral replication in immune horses. In contrast, complete viral replication with high viral copy numbers and shedding of infectious viruses was characteristic for non-immune horses, together with low or absent EHV-1-specific neutralizing antibodies during viral replication. These data confirm that pre-existing mucosal IgG1 and IgG4/7 and rapid B-cell activation upon EHV-1 infection are essential for virus neutralization, regulation of viral replication, and mucosal immunity against EHV-1.IMPORTANCEEquine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion storms, and neurologic outbreaks known as equine herpes myeloencephalopathy (EHM). EHV-1 is transmitted with respiratory secretions by nose-to-nose contact or via fomites. The virus initially infects the epithelium of the upper respiratory tract (URT). Host-pathogen interactions and mucosal immunity at the viral entry site provide the first line of defense against the EHV-1. Robust mucosal immunity can be essential in protecting against EHV-1 and to reduce EHM outbreaks. It has previously been shown that immune horses do not establish cell-associated viremia, the prerequisite for EHM. Here, we demonstrate how mucosal antibodies can prevent the replication of EHV-1 at the epithelium of the URT and, thereby, the progression of the virus to the peripheral blood. The findings improve the mechanistic understanding of mucosal immunity against EHV-1 and can support the development of enhanced diagnostic tools, vaccines against EHM, and the management of EHV-1 outbreaks.