A novel alternative for pyrogen detection based on a transgenic cell line.

Pyrogen, often as a contaminant, is a key indicator affecting the safety of almost all parenteral drugs (including biologicals, chemicals, traditional Chinese medicines and medical devices). It has become a goal to completely replace the in vivo rabbit pyrogen test by using the in vitro pyrogen test based on the promoted 'reduction, replacement and refinement' principle, which has been highly considered by regulatory agencies from different countries. We used NF-κB, a central signalling molecule mediating inflammatory responses, as a pyrogenic marker and the monocyte line THP-1 transfected with a luciferase reporter gene regulated by NF-κB as an in vitro model to detect pyrogens by measuring the intensity of a fluorescence signal. Here, we show that this test can quantitatively and sensitively detect endotoxin (lipopolysaccharide from different strains) and nonendotoxin (lipoteichoic acid, zymosan, peptidoglycan, lectin and glucan), has good stability in terms of NF-κB activity and cell phenotypes at 39 cell passages and can be applied to detect pyrogens in biologicals (group A & C meningococcal polysaccharide vaccine; basiliximab; rabies vaccine (Vero cells) for human use, freeze-dried; Japanese encephalitis vaccine (Vero cells), inactivated; insulin aspart injection; human albumin; recombinant human erythropoietin injection (CHO Cell)). The within-laboratory reproducibility of the test in three independent laboratories was 85%, 80% and 80% and the interlaboratory reproducibility among laboratories was 83.3%, 95.6% and 86.7%. The sensitivity (true positive rate) and specificity (true negative rate) of the test were 89.9% and 90.9%, respectively. In summary, the test provides a novel alternative for pyrogen detection.

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.

mRNA cancer vaccines: Advances, trends and challenges.

Patients exhibit good tolerance to messenger ribonucleic acid (mRNA) vaccines, and the choice of encoded molecules is flexible and diverse. These vaccines can be engineered to express full-length antigens containing multiple epitopes without major histocompatibility complex (MHC) restriction, are relatively easy to control and can be rapidly mass produced. In 2021, the U.S. Food and Drug Administration (FDA) approved the first mRNA-based coronavirus disease 2019 (COVID-19) vaccine produced by Pfizer and BioNTech, which has generated enthusiasm for mRNA vaccine research and development. Based on the above characteristics and the development of mRNA vaccines, mRNA cancer vaccines have become a research hotspot and have undergone rapid development, especially in the last five years. This review analyzes the advances in mRNA cancer vaccines from various perspectives, including the selection and expression of antigens/targets, the application of vectors and adjuvants, different administration routes, and preclinical evaluation, to reflect the trends and challenges associated with these vaccines.

Development of an ELISA Assay for the Determination of SARS-CoV-2 Protein Subunit Vaccine Antigen Content.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein subunit vaccine is one of the mainstream technology platforms for the development of COVID-19 vaccines, and most R&D units use the receptor-binding domain (RBD) or spike (S) protein as the main target antigen. The complexity of vaccine design, sequence, and expression systems makes it urgent to establish common antigen assays to facilitate vaccine development. In this study, we report the development of a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the antigen content of SARS-CoV-2 protein subunit vaccines based on the United States Pharmacopeia <1220> and ICH (international conference on harmonization) Q14 and Q2 (R2) requirements. A monoclonal antibody (mAb), 20D8, was identified as the detection antibody based on its high RBD binding activity (EC50 = 8.4 ng/mL), broad-spectrum anti-variant neutralizing activity (EC50: 2.7−9.8 ng/mL for pseudovirus and EC50: 9.6−127 ng/mL for authentic virus), good in vivo protection, and a recognized linear RBD epitope (369−379 aa). A porcine anti-RBD polyclonal antibody was selected as the coating antibody. Assay performance met the requirements of the analytical target profile with an accuracy and precision of ≥90% and adequate specificity. Within the specification range of 70−143%, the method capability index was >0.96; the misjudgment probability was <0.39%. The method successfully detected SARS-CoV-2 protein subunit vaccine antigens (RBD or S protein sequences in Alpha, Beta, Gamma, or Delta variants) obtained from five different manufacturers. Thus, we present a new robust, reliable, and general method for measuring the antigenic content of SARS-CoV-2 protein subunit vaccines. In addition to currently marketed and emergency vaccines, it is suitable for vaccines in development containing antigens derived from pre-Omicron mutant strains.

In vitro activity of protegrin-1 and beta-defensin-1, alone and in combination with isoniazid, against Mycobacterium tuberculosis.

The antimicrobial peptide protegrin-1 (PG-1) inhibited the growth in vitro of drug-susceptible and multidrug-resistant Mycobacterium tuberculosis; a lower activity was shown by human beta-defensin-1 (HBD-1) against both strains. The combination of PG-1 or HBD-1 with isoniazid significantly reduced M. tuberculosis growth in comparison with the peptides or isoniazid alone.

Comparison of result judgment algorithm of test for interfering factors in the bacterial endotoxins test among Chinese, Japanese, European, American, and Indian pharmacopeias.

BACKGROUND: The bacterial endotoxins test (BET) is a method used to detect or quantify endotoxins (lipo-polysaccharide, LPS) and is widely used in the quality control of parenteral medicines/vaccines and clinical dialysis fluid. It is also used in the diagnosis of endotoxemia and in detection of environment air quality control. Although BET has been adopted by most pharmacopoeias, result judgment algorithms (RJAs) of the test for interfering factors in the BET still differ between certain pharmacopoeias. We have evaluated RJAs of the test for interfering factors for the revision of BET described in the Chinese Pharmacopoeia 2010 (CHP2010). METHODS: Original data from 1 748 samples were judged by RJAs of the Chinese Pharmacopoeia 2010, the Japanese Pharmacopoeia 2011 (JP2011), the European Pharmacopoeia 7.0 (EP7.0), the United States Pharmacopoeia 36 (USP36), and the Indian Pharmacopoeia 2010 (IP2010), respectively. A SAS software package was used in the statistical analysis. RESULTS: The results using CHP2010 and USP36, JP2011, EP7.0, and IP2010 had no significant difference (P = 0.7740). The results using CHP2010 of 1 748 samples showed that 132 samples (7.6%) required an additional step; nevertheless there was no such requirement when using the other pharmacopeias. The kappa value of two RJAs (CHP2010 and EP7.0) was 0.6900 (0.6297-0.7504) indicating that the CHP2010 and other pharmacopoeias have good consistency. CONCLUSIONS: The results using CHP2010 and USP36, JP2011, EP7.0, and IP2010 have different characteristics. CHP2010 method shows a good performance in Specificity, mistake diagnostic rate, agreement rate, predictive value for suspicious rate, and predictive value for passed rate. The CHP2010 method only had disadvantages in sensitivity compared with other pharmacopeias. We suggest that the Chinese pharmacopoeia interference test be revised in accordance with the USP36, JP2011, EP7.0, and IP2010 judgment model.

Comparison of temperature rise interpretations in the rabbit pyrogen test among Chinese, Japanese, European, and United States pharmacopeias and 2-2-2 theoretical models proposed by S. Hoffmann.

Although the rabbit pyrogen test is one of the crucial methods included in each pharmacopeia to evaluate the safety of parenteral medicine, the experimental procedures and pyrogen result judgment algorithms (PRJAs) are still greatly different from one another. In the first stage of testing, original data of 879 batches from a total of 2637 rabbits in our laboratory were judged by PRJAs in the Chinese Pharmacopoeia 2005 III, the Japanese Pharmacopoeia XIV, the Japanese Pharmacopoeia XV, the European Pharmacopeia 6.0, the United States Pharmacopoeia 32 NF27 and two theoretical models proposed by S. Hoffmann, respectively. The results were analyzed to evaluate the effects of various PRJAs. It was shown that: (i) the significant differences in the results judged by various pharmacopeias and Hoffmann's theoretical models were mainly due to the PRJAs and the great differences in PRJAs should be harmonized throughout the world based on balance of reducing animal use and guaranteeing the safety of medicines; (ii) it is better to use PRJAs that depend on the threshold of the sum of temperature rise of all tested rabbits than those that depend on the number of rabbits that are over the threshold of temperature rise of individual rabbit according to clinical proof and the experimental data; and (iii) the PRJA of the Japanese Pharmacopoeia XV has obvious advantages when the total suspicious rate of samples was less than 10%. Additionally, a new PRJA designed for reducing the additional experiment stages and animal consumption is promoted for evaluation.

The extra cytoplasmic function sigma factor sigma(E) is essential for Mycobacterium tuberculosis virulence in mice.

The virulence of a Mycobacterium tuberculosis H37Rv sigE mutant was studied in immunodeficient and immunocompetent mice. The mutant was strongly attenuated in both animal models and induced formation of granulomas with different characteristics than those induced by the wild-type strain.

Involvement of the fadD33 gene in the growth of Mycobacterium tuberculosis in the liver of BALB/c mice.

The potential pathogenic role of Mycobacterium tuberculosis H37Rv fadD33, a gene encoding an acyl-CoA synthase that is underexpressed in the attenuated strain H37Ra, was investigated. In a first approach, fadD33 was cloned and expressed in strain H37Ra to restore gene expression and fadD33-complemented bacteria were used to investigate whether fadD33 might confer any growth advantage to M. tuberculosis H37Ra in an infection model of BALB/c mice. No differences were found in the growth rates of M. tuberculosis H37Rv, H37Ra and fadD33-complemented H37Ra in the lungs and spleen. In contrast, in the liver, where the attenuated strain H37Ra showed impaired growth compared to the virulent strain H37Rv, complementation of the attenuated strain H37Ra with fadD33 restored bacterial replication. In a further approach, the fadD33 gene of strain H37Rv was disrupted by allelic exchange mutagenesis and the virulence of the mutant strain was tested by mouse infection. It was found that disruption of fadD33 decreased M. tuberculosis H37Rv growth in the liver, but not in the lungs or spleen, and complementation of the fadD33-disrupted mutant with fadD33 restored bacterial replication in the liver, but did not affect replication in the lungs and spleen. These findings suggest that fadD33 plays a role in M. tuberculosis virulence by supporting bacterial growth in the liver.

Activities of moxifloxacin alone and in combination with other antimicrobial agents against multidrug-resistant Mycobacterium tuberculosis infection in BALB/c mice.

The activity of moxifloxacin was enhanced by the addition of ethionamide but not by that of cycloserine, thiacetazone, capreomycin, para-aminosalicylic acid, or linezolid in BALB/c mice infected with a strain of Mycobacterium tuberculosis resistant to isoniazid, rifampin, and six other drugs. These observations are important for the therapy of multidrug-resistant tuberculosis.

Exposure of BALB/c mice to low doses of Mycobacterium avium increases resistance to a subsequent high-dose infection.

BALB/c mice exposed intranasally (i.n.), intradermally (i.d.) or intraperitoneally (i.p.) to low doses of Mycobacterium avium (20 c.f.u. at three different times two weeks apart) showed an increased resistance to a subsequent high-dose (10(5) c.f.u.) infection. I.n.-exposed mice had few mycobacteria in the tissues (>100 c.f.u.) and showed an expansion of CD4(+) T cells associated with overproduction of IL-12 and IFN-gamma, but not IL-4 and IgG antibodies. Parenterally (i.p. and i.d.) exposed animals showed c.f.u. numbers higher than i.n.-exposed mice, together with overproduction of IL-12, IFN-gamma and IL-4 in the case of i.p.-exposed mice, and of IL-12, IFN-gamma and IgG2a and IgG1 antibodies in the case of i.d.-exposed mice. Low-dose exposures were not contained by athymic BALB/c nude mice; however, naive nude mice reconstituted with i.n.-primed CD4(+) T cells of BALB/c mice were protected against high-dose infection, indicating that CD4(+) T cells are essential to control even low-dose infections by M. avium. Overall, these data suggest that continuous i.n. exposure to M. avium doses commonly found in the environment may play a role in determining the natural resistance of normal hosts against this organism.