Development and application of a high-sensitivity immunochromatographic test strip for detecting pseudorabies virus.

Introduction: Pseudorabies (PR) is a multi-animal comorbid disease caused by pseudorabies virus (PRV), which are naturally found in pigs. At the end of 2011, the emergence of PRV variant strains in many provinces in China had caused huge economic losses to pig farms. Rapid detection diagnosis of pigs infected with the PRV variant helps prevent outbreaks of PR. The immunochromatography test strip with colloidal gold nanoparticles is often used in clinical testing due to its low cost and high throughput. Methods: This study was designed to produce monoclonal antibodies targeting PRV through immunization of mice using the eukaryotic system to express the gE glycoprotein. Subsequently, paired monoclonal antibodies were screened based on their sensitivity and specificity for use in the preparation of test strips. Results and discussion: The strip prepared in this study was highly specific, only PRV was detected, and there was no cross-reactivity with glycoprotein gB, glycoprotein gC, glycoprotein gD, and glycoprotein gE of herpes simplex virus and varicellazoster virus, porcine epidemic diarrhea virus, Senecavirus A, classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine parvovirus. Moreover, it demonstrated high sensitivity with a detection limit of 1.336 × 103 copies/µL (the number of viral genome copies per microliter); the coincidence rate with the RT-PCR detection method was 96.4%. The strip developed by our laboratory provides an effective method for monitoring PRV infection and controlling of PR vaccine quality.

Research progress on the quality control of mRNA vaccines.

INTRODUCTION: The mRNA vaccine technologies have progressed rapidly in recent years. The COVID-19 pandemic has accelerated the application of mRNA vaccines, with research and development and clinical trials underway for many vaccines. Application of the quality by design (QbD) framework to mRNA vaccine development and establishing standardized quality control protocols for mRNA vaccines are essential for the continued development of high-quality mRNA vaccines. AREAS COVERED: mRNA vaccines include linear mRNA, self-amplifying mRNA, and circular RNA vaccines. This article summarizes the progress of research on quality control of these three types of vaccines and presents associated challenges and considerations. EXPERT OPINION: Although there has been rapid progress in research on linear mRNA vaccines, their degradation patterns remain unclear. In addition, standardized assays for key impurities, such as residual dsRNA and T7 RNA polymerase, are still lacking. For self-amplifying mRNA vaccines, a key focus should be control of stability in vivo and in vitro. For circular RNA vaccines, standardized assays, and reference standards for determining degree of circularization should be established and optimized.

Establishment of the First National Standard for Neutralizing Antibodies against SARS-CoV-2 XBB Variants.

Neutralizing antibodies (NtAbs) against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are indicators of vaccine efficacy that enable immunity surveillance. However, the rapid mutation of SARS-CoV-2 variants prevents the timely establishment of standards required for effective XBB vaccine evaluation. Therefore, we prepared four candidate standards (No. 11, No. 44, No. 22, and No. 33) using plasma, purified immunoglobulin, and a broad-spectrum neutralizing monoclonal antibody. Collaborative calibration was conducted across nine Chinese laboratories using neutralization methods against 11 strains containing the XBB and BA.2.86 sublineages. This study demonstrated the reduced neutralization potency of the first International Standard antibodies to SARS-CoV-2 variants of concern against XBB variants. No. 44 displayed broad-spectrum neutralizing activity against XBB sublineages, effectively reduced interlaboratory variability for nearly all XBB variants, and effectively minimized the geometric mean titer (GMT) difference between the live and pseudotyped virus. No. 22 showed a broader spectrum and higher neutralizing activity against all strains but failed to reduce interlaboratory variability. Thus, No. 44 was approved as a National Standard for NtAbs against XBB variants, providing a unified NtAb measurement standard for XBB variants for the first time. Moreover, No. 22 was approved as a national reference reagent for NtAbs against SARS-CoV-2, offering a broad-spectrum activity reference for current and potentially emerging variants.

Progress and challenges in the clinical evaluation of immune responses to respiratory mucosal vaccines.

INTRODUCTION: Following the coronavirus disease pandemic, respiratory mucosal vaccines that elicit both mucosal and systemic immune responses have garnered increasing attention. However, human physiological characteristics pose significant challenges in the evaluation of mucosal immunity, which directly impedes the development and application of respiratory mucosal vaccines. AREAS COVERED: This study summarizes the characteristics of immune responses in the respiratory mucosa and reviews the current status and challenges in evaluating immune response to respiratory mucosal vaccines. EXPERT OPINION: Secretory Immunoglobulin A (S-IgA) is a major effector molecule at mucosal sites and a commonly used indicator for evaluating respiratory mucosal vaccines. However, the unique physiological structure of the respiratory tract pose significant challenges for the clinical collection and detection of S-IgA. Therefore, it is imperative to develop a sampling method with high collection efficiency and acceptance, a sensitive detection method, reference materials for mucosal antibodies, and to establish a threshold for S-IgA that correlates with clinical protection. Sample collection is even more challenging when evaluating mucosal cell immunity. Therefore, a mucosal cell sampling method with high operability and high tolerance should be established. Targets of the circulatory system capable of reflecting mucosal cellular immunity should also be explored.

A Cocktail of Lipid Nanoparticle-mRNA Vaccines Broaden Immune Responses against ß-Coronaviruses in a Murine Model.

Severe acute respiratory syndrome (SARS)-coronavirus (CoV), Middle Eastern respiratory syndrome (MERS)-CoV, and SARS-CoV-2 have seriously threatened human life in the 21st century. Emerging and re-emerging ß-coronaviruses after the coronavirus disease 2019 (COVID-19) epidemic remain possible highly pathogenic agents that can endanger human health. Thus, pan-ß-coronavirus vaccine strategies to combat the upcoming dangers are urgently needed. In this study, four LNP-mRNA vaccines, named O, D, S, and M, targeting the spike protein of SARS-CoV-2 Omicron, Delta, SARS-CoV, and MERS-CoV, respectively, were synthesized and characterized for purity and integrity. All four LNP-mRNAs induced effective cellular and humoral immune responses against the corresponding spike protein antigens in mice. Furthermore, LNP-mRNA S and D induced neutralizing antibodies against SARS-CoV and SARS-CoV-2, which failed to cross-react with MERS-CoV. Subsequent evaluation of sequential and cocktail immunizations with LNP-mRNA O, D, S, and M effectively elicited broad immunity against SARS-CoV-2 variants, SARS-CoV, and MERS-CoV. A direct comparison of the sequential with cocktail regimens indicated that the cocktail vaccination strategy induced more potent neutralizing antibodies and T-cell responses against heterotypic viruses as well as broader antibody activity against pan-ß-coronaviruses. Overall, these results present a potential pan-ß-coronavirus vaccine strategy for improved preparedness prior to future coronavirus threats.

A screening study on the detection strain of Coxsackievirus A6: the key to evaluating neutralizing antibodies in vaccines.

The increasing incidence of diseases caused by Coxsackievirus A6 (CV-A6) and the presence of various mutants in the population present significant public health challenges. Given the concurrent development of multiple vaccines in China, it is challenging to objectively and accurately evaluate the level of neutralizing antibody response to different vaccines. The choice of the detection strain is a crucial factor that influences the detection of neutralizing antibodies. In this study, the National Institutes for Food and Drug Control collected a prototype strain (Gdula), one subgenotype D1, as well as 13 CV-A6 candidate vaccine strains and candidate detection strains (subgenotype D3) from various institutions and manufacturers involved in research and development. We evaluated cross-neutralization activity using plasma from naturally infected adults (n = 30) and serum from rats immunized with the aforementioned CV-A6 strains. Although there were differences between the geometric mean titer (GMT) ranges of human plasma and murine sera, the overall trends were similar. A significant effect of each strain on the neutralizing antibody test (MAX/MIN 48.0 ∼16410.3) was observed. Among all strains, neutralization of the S112 strain by 15 different sera resulted in higher neutralizing antibody titers (GMTS112 = 132.0) and more consistent responses across different genotypic immune sera (MAX/MIN = 48.0). Therefore, S112 may serve as a detection strain for NtAb testing in various vaccines, minimizing bias and making it suitable for evaluating the immunogenicity of the CV-A6 vaccine.

Amplifying mRNA vaccines: potential versatile magicians for oncotherapy.

Cancer vaccines drive the activation and proliferation of tumor-reactive immune cells, thereby eliciting tumor-specific immunity that kills tumor cells. Accordingly, they possess immense potential in cancer treatment. However, such vaccines are also faced with challenges related to their design and considerable differences among individual tumors. The success of messenger RNA (mRNA) vaccines against coronavirus disease 2019 has prompted the application of mRNA vaccine technology platforms to the field of oncotherapy. These platforms include linear, circular, and amplifying mRNA vaccines. In particular, amplifying mRNA vaccines are characterized by high-level and prolonged antigen gene expression at low doses. They can also stimulate specific cellular immunity, making them highly promising in cancer vaccine research. In this review, we summarize the research progress in amplifying mRNA vaccines and provide an outlook of their prospects and future directions in oncotherapy.

Non-small cell lung cancers (NSCLCs) oncolysis using coxsackievirus B5 and synergistic DNA-damage response inhibitors.

With the continuous in-depth study of the interaction mechanism between viruses and hosts, the virus has become a promising tool in cancer treatment. In fact, many oncolytic viruses with selectivity and effectiveness have been used in cancer therapy. Human enterovirus is one of the most convenient sources to generate oncolytic viruses, however, the high seroprevalence of some enteroviruses limits its application which urges to exploit more oncolytic enteroviruses. In this study, coxsackievirus B5/Faulkner (CV-B5/F) was screened for its potential oncolytic effect against non-small cell lung cancers (NSCLCs) through inducing apoptosis and autophagy. For refractory NSCLCs, DNA-dependent protein kinase (DNA-PK) or ataxia telangiectasia mutated protein (ATM) inhibitors can synergize with CV-B5/F to promote refractory cell death. Here, we showed that viral infection triggered endoplasmic reticulum (ER) stress-related pro-apoptosis and autophagy signals, whereas repair for double-stranded DNA breaks (DSBs) contributed to cell survival which can be antagonized by inhibitor-induced cell death, manifesting exacerbated DSBs, apoptosis, and autophagy. Mechanistically, PERK pathway was activated by the combination of CV-B5/F and inhibitor, and the irreversible ER stress-induced exacerbated cell death. Furthermore, the degradation of activated STING by ERphagy promoted viral replication. Meanwhile, no treatment-related deaths due to CV-B5/F and/or inhibitors occurred. Conclusively, our study identifies an oncolytic CV-B5/F and the synergistic effects of inhibitors of DNA-PK or ATM, which is a potential therapy for NSCLCs.

A Novel Targeted RIG-I Receptor 5'Triphosphate Double Strain RNA-Based Adjuvant Significantly Improves the Immunogenicity of the SARS-CoV-2 Delta-Omicron Chimeric RBD-Dimer Recombinant Protein Vaccine.

The rapid mutation and spread of SARS-CoV-2 variants recently, especially through the emerging variants Omicron BA5, BF7, XBB and BQ1, necessitate the development of universal vaccines to provide broad spectrum protection against variants. For the SARS-CoV-2 universal recombinant protein vaccines, an effective approach is necessary to design broad-spectrum antigens and combine them with novel adjuvants that can induce high immunogenicity. In this study, we designed a novel targeted retinoic acid-inducible gene-I (RIG-I) receptor 5'triphosphate double strain RNA (5'PPP dsRNA)-based vaccine adjuvant (named AT149) and combined it with the SARS-CoV-2 Delta and Omicron chimeric RBD-dimer recombinant protein (D-O RBD) to immunize mice. The results showed that AT149 activated the P65 NF-κB signaling pathway, which subsequently activated the interferon signal pathway by targeting the RIG-I receptor. The D-O RBD + AT149 and D-O RBD + aluminum hydroxide adjuvant (Al) + AT149 groups showed elevated levels of neutralizing antibodies against the authentic Delta variant, and Omicron subvariants, BA1, BA5, and BF7, pseudovirus BQ1.1, and XBB compared with D-O RBD + Al and D-O RBD + Al + CpG7909/Poly (I:C) groups at 14 d after the second immunization, respectively. In addition, D-O RBD + AT149 and D-O RBD + Al + AT149 groups presented higher levels of the T-cell-secreted IFN-γ immune response. Overall, we designed a novel targeted RIG-I receptor 5'PPP dsRNA-based vaccine adjuvant to significantly improve the immunogenicity and broad spectrum of the SARS-CoV-2 recombinant protein vaccine.

Establishment of national standard for anti-SARS-Cov-2 neutralizing antibody in China: The first National Standard calibration traceability to the WHO International Standard.

Neutralizing antibody (NtAb) levels are key indicators in the development and evaluation of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines. Establishing a unified and reliable WHO International Standard (IS) for NtAb is crucial for the calibration and harmonization of NtAb detection assays. National and other WHO secondary standards are key links in the transfer of IS to working standards but are often overlooked. The Chinese National Standard (NS) and WHO IS were developed by China and WHO in September and December 2020, respectively, the application of which prompted and coordinated sero-detection of vaccine and therapy globally. Currently, a second-generation Chinese NS is urgently required owing to the depletion of stocks and need for calibration to the WHO IS. The Chinese National Institutes for Food and Drug Control (NIFDC) developed two candidate NSs (samples 33 and 66-99) traced to the IS according to the WHO manual for the establishment of national secondary standards through a collaborative study of nine experienced labs. Either NS candidate can reduce the systematic error among different laboratories and the difference between the live virus neutralization (Neut) and pseudovirus neutralization (PsN) methods, ensuring the accuracy and comparability of NtAb test results among multiple labs and methods, especially for samples 66-99. At present, samples 66-99 have been approved as the second-generation NS, which is the first NS calibrated tracing to the IS with 580 (460-740) International Units (IU)/mL and 580 (520-640) IU/mL by Neut and PsN, respectively. The use of standards improves the reliability and comparability of NtAb detection, ensuring the continuity of the use of the IS unitage, which effectively promotes the development and application of SARS-CoV-2 vaccines in China.

Research Advances on the Stability of mRNA Vaccines.

Compared to other vaccines, the inherent properties of messenger RNA (mRNA) vaccines and their interaction with lipid nanoparticles make them considerably unstable throughout their life cycles, impacting their effectiveness and global accessibility. It is imperative to improve mRNA vaccine stability and investigate the factors influencing stability. Since mRNA structure, excipients, lipid nanoparticle (LNP) delivery systems, and manufacturing processes are the primary factors affecting mRNA vaccine stability, optimizing mRNA structure and screening excipients can effectively improve mRNA vaccine stability. Moreover, improving manufacturing processes could also prepare thermally stable mRNA vaccines with safety and efficacy. Here, we review the regulatory guidance associated with mRNA vaccine stability, summarize key factors affecting mRNA vaccine stability, and propose a possible research path to improve mRNA vaccine stability.

Pseudotyped Viruses for Enterovirus.

Using a non-pathogenic pseudotyped virus as a surrogate for a wide-type virus in scientific research complies with the recent requirements for biosafety. Enterovirus (EV) contains many species of viruses, which are a type of nonenveloped virus. The preparation of its corresponding pseudotyped virus often needs customized construction compared to some enveloped viruses. This article describes the procedures and challenges in the construction of pseudotyped virus for enterovirus (pseudotyped enterovirus, EVpv) and also introduces the application of EVpv in basic virological research, serological monitoring, and the detection of neutralizing antibody (NtAb).

Research progress on substitution of in vivo method(s) by in vitro method(s) for human vaccine potency assays.

INTRODUCTION: Potency is a critical quality attribute for controlling quality consistency and relevant biological properties of vaccines. Owing to the high demand for animals, lengthy operations and high variability of in vivo methods, in vitro alternatives for human vaccine potency assays are extensively developed. AREAS COVERED: Herein, in vivo and in vitro methods for potency assays of previously licensed human vaccines were sorted, followed by a brief description of the background for substituting in vivo methods with in vitro alternatives. Based on the analysis of current research on the substitution of vaccine potency assays, barriers and suggestions for substituting were proposed. EXPERT OPINION: Owing to the variability of in vivo methods, the correlation between in vivo and in vitro methods may be low. One or more in vitro method(s) that determine the vaccine antigen content and functions, should be established. Since the substitution involves with the change of critical quality attributes and specifications, the specifications of in vitro methods should be appropriately set to maintain the efficacy of vaccines. For novel vaccines in research and development, in vitro methods for monitoring the consistency and relevant biological properties, should be established based on reflecting the immunogenicity of vaccines.

Establishment of the 1st Chinese national standard for CA6 neutralizing antibody.

Coxsackievirus A6 (CA6) is one of the major causative agents of herpangina and hand-foot-mouth disease (HFMD). Since 2008, CA6 has circulated widely around the world. Especially in Asia-Pacific region CA6 had even replaced enterovirus A71 (EV71) and coxsackievirus A16 (CA16) as the main prevalent strain of HFMD. In the recent 10 years, monovalent and multivalent vaccines against CA6 have been researched and developed by manufacturers from China, Korea, and the USA. The neutralizing antibody titer is a key indicator for accurately evaluating immunogenicity of vaccine. However, so far, the World Health Organization international standard for CA6 neutralizing antibody has not been available. In order to meet the needs of evaluating the immunogenicity of vaccines against CA6, the first Chinese national standard for CA6 neutralizing antibody was established, which was conducted to ensure that methods used to measure the neutralizing antibody titers against CA6 are accurate, reliable, and comparable. Three lyophilized candidate standards (29#, 39# and 44#) were produced with 0.40 ml/vial from plasma samples donated by healthy individuals. The collaborative study showed that the 29# candidate standard could effectively minimize the variability in neutralization titers between labs and across challenging viruses of different genotypes (A, D1, and D3). Therefore, the 29# candidate sample was established as the first Chinese national standard for CA6 neutralizing antibody test. This standard has good long-term stability and was assigned a potency of 150 units per milliliter (U/ml) of CA6 neutralizing antibody. It will contribute to ensure uniformity of potency or activity of vaccines and potentially therapeutic antibody preparations.

Heterologous Omicron-adapted vaccine as a secondary booster promotes neutralizing antibodies against Omicron and its sub-lineages in mice.

Over one billion people have received 2-3 dosages of an inactivated COVID-19 vaccine for basic immunization. Whether a booster dose should be delivered to protect against the Omicron variant and its sub-lineages, remains controversial. Here, we tested different vaccine platforms targeting the ancestral or Omicron strain as a secondary booster of the ancestral inactivated vaccine in mice. We found that the Omicron-adapted inactivated viral vaccine promoted a neutralizing antibody response against Omicron in mice. Furthermore, heterologous immunization with COVID-19 vaccines based on different platforms remarkably elevated the levels of cross- neutralizing antibody against Omicron and its sub-lineages. Omicron-adapted vaccines based on heterologous platforms should be prioritized in future vaccination strategies to control COVID-19.

Non-neutralizing monoclonal antibody targeting VP2 EF loop of Coxsackievirus A16 can protect mice from lethal attack via Fc-dependent effector mechanism.

Coxsackievirus A16 (CA16), a main causative agent of hand, foot, and mouth disease (HFMD), has become a serious public health concern in the Asia-Pacific region. Here, we generated an anti-CA16 monoclonal antibody, DMA2017, derived from an epidemic strain CA16. Surprisingly, although DMA2017 could not neutralize the original and circulating CA16 strains in vitro, the passive transfer of DMA2017 (10 µg/g) could protect suckling mice from a lethal challenge with CA16 in vivo. Then, we confirmed the protective effect of DMA2017 relies on the Fc-dependent effector functions, such as antibody-dependent cellular cytotoxicity (ADCC). The linear epitope of DMA2017 was mapped by phage display technique to a conserved patch spanning residues 143-148 (NSHPPY) of the VP2 EF-loop of CA16. DMA2017 could inhibit the binding of the antibodies present in the sera of naturally infected children to CA16, indicating that the epitope of DMA2017 is immunodominant for CA16. Our results confirm, for the first time, that a potential preventive and therapeutic effect could be mediated by a non-neutralizing antibody elicited against CA16. These findings bring a hitherto understudied protective role of non-neutralizing antibodies during viral infections into the spotlight and provide a new perspective on the design and evaluation of CA16 vaccines.

Mixed formulation of mRNA and protein-based COVID-19 vaccines triggered superior neutralizing antibody responses.

Integrating different types of vaccines into a singular immunization regimen is an effective and accessible approach to strengthen and broaden the immunogenicity of existing coronavirus disease 2019 (COVID-19) vaccine candidates. To optimize the immunization strategy of the novel mRNA-based vaccine and recombinant protein subunit vaccine that attracted much attention in COVID-19 vaccine development, we evaluated the immunogenicity of different combined regimens with the mRNA vaccine (RNA-RBD) and protein subunit vaccine (PS-RBD) in mice. Compared with homologous immunization of RNA-RBD or PS-RBD, heterologous prime-boost strategies for mRNA and protein subunit vaccines failed to simultaneously enhance neutralizing antibody (NAb) and Th1 cellular response in this study, showing modestly higher serum neutralizing activity and antibody-dependent cell-mediated cytotoxicity for "PS-RBD prime, RNA-RBD boost" and robust Th1 type cellular response for "RNA-RBD prime, PS-RBD boost". Interestingly, immunizing the mice with the mixed formulation of the two aforementioned vaccines in various proportions further significantly enhanced the NAb responses against ancestral, Delta, and Omicron strains and manifested increased Th1-type responses, suggesting that a mixed formulation of mRNA and protein vaccines might be a more prospective vaccination strategy. This study provides basic research data on the combined vaccination strategies of mRNA and protein-based COVID-19 vaccines.

A Short 5'triphosphate RNA nCoV-L Induces a Broad-Spectrum Antiviral Response by Activating RIG-I.

Small molecular nucleic acid drugs produce antiviral effects by activating pattern recognition receptors (PRRs). In this study, a small molecular nucleotide containing 5'triphosphoric acid (5'PPP) and possessing a double-stranded structure was designed and named nCoV-L. nCoV-L was found to specifically activate RIG-I, induce interferon responses, and inhibit duplication of four RNA viruses (Human enterovirus 71, Human poliovirus 1, Human coxsackievirus B5 and Influenza A virus) in cells. In vivo, nCoV-L quickly induced interferon responses and protected BALB/c suckling mice from a lethal dose of the enterovirus 71. Additionally, prophylactic administration of nCoV-L was found to reduce mouse death and relieve morbidity symptoms in a K18-hACE2 mouse lethal model of SARS-CoV-2. In summary, these findings indicate that nCoV-L activates RIG-I and quickly induces effective antiviral signals. Thus, it has potential as a broad-spectrum antiviral drug.