Replacing the in vivo toxin challenge test with an in vitro assay for assessment of potency for diphtheria toxoid containing vaccines.

Replacement of the potency tests for diphtheria vaccines is a high priority for the international initiative to reduce, refine, and replace animal use in vaccine testing. Diphtheria toxoid containing vaccine products marketed in the US currently require potency testing by the United States Public Health Service (USPHS) test, which includes an in vivo passive protection test with a diphtheria toxin challenge. Here we describe an in vitro Diphtheria Vero Cell (DVC) assay which combines the immunization approach from the USPHS test and the use of a cell based neutralization assay for serological testing of vaccine potency. The DVC assay reduces the overall number of animals used compared to other serological potency tests and eliminates the in vivo toxin challenge used in the US test. The DVC assay can be used to test vaccine products with a low or high diphtheria toxoid dose. It has been optimized and validated for use in a quality control testing environment. Results demonstrate similar sera antibody unitage as well as agreement between the serum neutralization values determined using the USPHS test and the DVC assay and thus support the use of the DVC assay for routine and stability testing for diphtheria toxoid containing vaccine products.

Detection of avian retroviruses in vaccines by amplification on DF-1 cells with immunostaining and fluorescent product-enhanced reverse transcriptase endpoint methods.

In order to ensure the safety of vaccines produced on avian cells, rigorous testing for the absence of avian retroviruses must be performed. Current methods used to detect avian retroviruses often exhibit a high invalid-test/false-positive rate, rely on hard-to-secure reagents, and/or have readouts that are difficult to standardize. Herein, we describe the development and validation of two consistent and sensitive methods for the detection of avian retroviruses in vaccines: viral amplification on DF-1 cells followed by immunostaining for the detection of avian leukosis virus (ALV) and viral amplification on DF-1 cells followed by fluorescent product-enhanced reverse transcriptase (F-PERT) for the detection of all avian retroviruses. Both assays share an infectivity stage on DF-1 cells followed by a different endpoint readout depending on the retrovirus to be detected. Validation studies demonstrated a limit of detection of one 50% cell culture infectious dose (CCID(50))/ml for retrovirus in a 30-ml test inoculum volume for both methods, which was as sensitive as a classical method used in the vaccine industry, namely, viral amplification on primary chicken embryo fibroblasts followed by the complement fixation test for avian leukosis virus (COFAL). Furthermore, viral amplification on DF-1 cells followed by either immunostaining or F-PERT demonstrated a sensitivity that exceeds the regulatory requirements for detection of ALV strains. A head-to-head comparison of the two endpoint methods showed that viral amplification on DF-1 cells followed by F-PERT is a suitable method to be used as a stand-alone test to ensure that vaccine preparations are free from infectious avian retroviruses.

Evaluation of infectious titer in a candidate HSV type 2 vaccine by a quantitative molecular approach.

BACKGROUND: One of the critical tasks in analytical testing is to monitor and assign the infectivity or potency of viral based vaccines from process development to production of final clinical lots. In this study, a high throughput RT-qPCR based approach was developed to evaluate the infectious titre in a replication-defective HSV-2 candidate vaccine, called HSV529. This assay is a combination of viral propagation and quantitative RT-PCR which measures the amount of RNA in infected cells after incubation with test samples. RESULTS: The relative infectious titre of HSV529 candidate vaccine was determined by a RT-qPCR method targeting HSV-2 gD2 gene. The data were analyzed using the parallel-line analysis as described in the European Pharmacopoeia 8th edition. The stability of HSV529 test samples were also investigated in a concordance study between RT-qPCR infectivity assay and a classical plaque assays. A suitable correlation was determined between both assays using an identical sample set in both assays. The RT-qPCR infectivity assay was further characterized by evaluating the intermediate precision and accuracy. The coefficient of variation from the six independent assays was less than 10%. The accuracy of each of the assay was also evaluated in the range of 92.91% to 120.57%. CONCLUSIONS: Our data demonstrate that the developed RT-qPCR infectivity assay is a rapid high throughput approach to quantify the infectious titer or potency of live attenuated or defective viral-based vaccines, an attribute which is associated with product quality.

Application of xCELLigence real-time cell analysis to the microplate assay for pertussis toxin induced clustering in CHO cells.

The microplate assay with Chinese Hamster Ovary (CHO) cells is currently used as a safety test to monitor the residual pertussis toxin (PT) amount in acellular pertussis antigens prior to vaccine formulation. The assay is based on the findings that the exposure of CHO cells to PT results in a concentration-dependent clustering response which can be used to estimate the amount of PT in a sample preparation. A major challenge with the current CHO cell assay methodology is that scoring of PT-induced clustering is dependent on subjective operator visual assessment using light microscopy. In this work, we have explored the feasibility of replacing the microscopy readout for the CHO cell assay with the xCELLigence Real-Time Cell Analysis system (ACEA BioSciences, a part of Agilent). The xCELLigence equipment is designed to monitor cell adhesion and growth. The electrical impedance generated from cell attachment and proliferation is quantified via gold electrodes at the bottom of the cell culture plate wells, which is then translated into a unitless readout called cell index. Results showed significant decrease in the cell index readouts of CHO cells exposed to PT compared to the cell index of unexposed CHO cells. Similar endpoint concentrations were obtained when the PT reference standard was titrated with either xCELLigence or microscopy. Testing genetically detoxified pertussis samples unspiked or spiked with PT further supported the sensitivity and reproducibility of the xCELLigence assay in comparison with the conventional microscopy assay. In conclusion, the xCELLigence RTCA system offers an alternative automated and higher throughput method for evaluating PT-induced clustering in CHO cells.

Sensitivity and breadth of detection of high-throughput sequencing for adventitious virus detection.

High-throughput sequencing (HTS) is capable of broad virus detection encompassing both known and unknown adventitious viruses in a variety of sample matrices. We describe the development of a general-purpose HTS-based method for the detection of adventitious viruses. Performance was evaluated using 16 viruses equivalent to well-characterized National Institutes of Health (NIH) virus stocks and another six viruses of interest. A viral vaccine crude harvest and a cell substrate matrix were spiked with 22 viruses. Specificity was demonstrated for all 22 viruses at the species level. Our method was capable of detecting and identifying adventitious viruses spiked at 104 genome copies per milliliter in a viral vaccine crude harvest and 0.01 viral genome copies spiked per cell in a cell substrate matrix. Moreover, 9 of the 11 NIH model viruses with published in vivo data were detected by HTS with an equivalent or better sensitivity (in a viral vaccine crude harvest). Our general-purpose HTS method is unbiased and highly sensitive for the detection of adventitious viruses, and has a large breadth of detection, which may obviate the need to perform in vivo testing.

An in vitro functional assay to measure the biological activity of TB vaccine candidate H4-IC31.

Potency assays for vaccine products are an important regulatory requirement, and are used to assess product quality and consistency prior to lot release for clinical testing. Ideally they measure an established correlate of efficacy or protection. In cases where there is no known correlate of protection, however, a functional assay that measures a biological response to a vaccine can be applied as a potency assay. Here we describe an in vitro assay which quantitatively measures human T cell activation as a biological response to the TB vaccine candidate H4-IC31. The Cytokine Secretion Assay (CSA) is based on the ability of peripheral blood mononuclear cells (PBMCs) from a Bacillus Calmette-Guérin (BCG)-vaccinated human donor to process and respond to H4-IC31. The ability of H4-IC31 to stimulate a cellular immune response is measured through the quantification of secreted IFNγ and is reported as relative stimulatory activity (RSA) compared to an in-house reference standard. The CSA is specific to the H4-IC31 vaccine, determines the RSA of H4-IC31 in the range of 50% to 150% of the reference standard, and is stability indicating as it detects differences in RSA between intact and heat treated H4-IC31. Although the CSA does not provide a link to clinical efficacy, it fulfills the critical requirements for a biological potency test to assess TB vaccine candidates and can be used along with biochemical and immunochemical assays to define a product profile during clinical development, while eliminating the use of animals for product testing.

Selection and evaluation of an efficient method for the recovery of viral nucleic acids from complex biologicals [corrected].

There is a need for a broad and efficient testing strategy for the detection of both known and novel viral adventitious agents in vaccines and biologicals. High-throughput sequencing (HTS) is an approach for such testing; however, an optimized testing method is one with a sample-processing pipeline that can help detect any viral adventitious agent that may be present. In this study, 11 commercial methods were assessed for efficient extraction of nucleic acids from a panel of viruses. An extraction strategy with two parallel arms, consisting of both the Invitrogen PureLink™ Virus RNA/DNA kit for total nucleic acid extraction and the Wako DNA Extractor® kit with an RNase A digestion for enrichment of double-stranded nucleic acid, was selected as the strategy for the extraction of all viral nucleic acid types (ssRNA, dsRNA, and dsDNA). Downstream processes, such as double-strand DNA synthesis and whole-genome amplification (WGA), were also assessed for the retrieval of viral sequences. Double-stranded DNA synthesis yielded larger numbers of viral reads, whereas WGA exhibited a strong bias toward amplification of double-stranded DNA, including host cellular DNA. The final sample-processing strategy consisted of the dual extraction approach followed by double-stranded DNA synthesis, which yielded a viral population with increased detection of some viruses by 8600-fold. Here we describe an efficient extraction procedure to support viral adventitious agent detection in cell substrates used for biological products using HTS.

Erratum: Author Correction: Selection and evaluation of an efficient method for the recovery of viral nucleic acids from complex biologicals.

[This corrects the article DOI: 10.1038/s41541-018-0067-3.].

Flow cytometry: An efficient method for antigenicity measurement and particle characterization on an adjuvanted vaccine candidate H4-IC31 for tuberculosis.

We have developed an accurate, precise and stability-indicating flow cytometry (FC) based assay to directly measure antigenicity of H4 protein (also known as HyVac4) in a vaccine formulation of H4-IC31, without desorbing the H4 protein from the IC31 adjuvant. This method involves immuno-staining of H4-IC31 complex with anti-H4 monoclonal antibodies (mAbs) followed by FC analysis. The assay is not only able to consistently measure H4 antigenicity levels in H4-IC31 stored under normal condition at 2-8°C, but also able to detect changes in H4 antigenicity after H4-IC31 undergoes heat stress or freeze-thawing. In addition, the FC method is able to characterize particle morphology while measuring antigenicity. The biological relevance of the changes in H4 antigenicity detected by the FC assay was supported by an in vitro cell based functional assay using human PBMCs to measure IFN-gamma (IFN-γ) secretion upon re-stimulation with H4-IC31. Our results show that the FC based antigenicity assay can efficiently monitor the biological and physicochemical properties of H4-IC31 and is an indicator for adjuvanted vaccine product stability.

In vitro assessment of biological activity and stability of the ALVAC-HIV vaccine.

The first evidence in humans that a safe and effective preventive vaccine for HIV is possible came from the phase III HIV clinical trial RV144 in Thailand. This trial was based on a prime/boost combination of a recombinant canarypox vaccine and two glycoprotein 120 proteins (ALVAC-HIV and AIDSVAX B/E). A pivotal phase IIb/III trial has recently commenced in the Republic of South Africa, for which the infectious titer assay was applied as the quantitative release test for the ALVAC-HIV vaccine. The infectious titer assay measures the ability of the vaccine vector to infect target permissive cells, but does not indicate if the vaccine transgenes are expressed. We have developed a high-throughput biological activity assay that provides results in agreement with the infectious titer assay. This assay uses flow cytometry to quantify expression of ALVAC-HIV encoded proteins gp120 and p24 in human cells. This transgene expression is detected by two cross-clade-reactive, biologically functional human anti-gp120 monoclonal antibodies isolated from clinical trial participants and a commercial mouse anti-p24 monoclonal antibody. The relative biological activity of the vaccine test sample is calculated by comparison of the test sample dose-response curve against that of a reference standard. We show that the novel biological activity assay is specific, accurate, precise, stability-indicating, and robust. The assay is being used for characterization of ALVAC-HIV (vCP2438) product, the efficacy of which is being evaluated in the pivotal phase IIb/III clinical trial HVTN702. The biological activity assay has the potential to indicate vaccine consistency and quality as a complement to the infectious titer assay.

New approaches for characterization of the genetic stability of vaccine cell lines.

The genetic stability of cell lines is a critical analytical attribute required to demonstrate the quality of cells over time. During cell passage, mutations can arise in the genomic DNA, potentially leading to changes in the final vaccine product. The identity and integrity of master cell banks, extended cell banks, complementing cell lines or recombinant cell lines expressing transgenes has to be tested throughout the production process by the vaccine manufacturer. Over the past few years, the traditional methods for evaluation of genetic stability have been replaced with molecular approaches including quantitative PCR, digital PCR and high throughput sequencing. However, these molecular-based approaches are used in research laboratories and not within a GMP-compliant environment. In this article, we briefly discuss some opportunities and challenges in characterization of the genetic stability of vaccine cell lines with these molecular-based approaches.

Need for new technologies for detection of adventitious agents in vaccines and other biological products.

From an industrial perspective, the conventional in vitro and in vivo assays used for detection of viral contaminants have shown their limitations, as illustrated by the unfortunate detection of porcine circovirus contamination in a licensed rotavirus vaccine. This contamination event illustrates the gaps within the existing adventitious agent strategy and the potential use of new broader molecular detection methods. This paper serves to summarize current testing approaches and challenges, along with opportunities for the use of these new technologies. LAY ABSTRACT: Testing of biological products is required to ensure the safety of patients. Recently, a licensed vaccine was found to be contaminated with a virus. This contamination did not cause a safety concern to the patients; however, it highlights the need for using new testing methods to control our biological products. This paper introduces the benefits of these new tests and outlines the challenges with the current tests.

Cataloguing the taxonomic origins of sequences from a heterogeneous sample using phylogenomics: applications in adventitious agent detection.

We have designed and implemented a software system, named PhyloID™, that can be used to detect putative adventitious agents in biological samples characterized by next-generation sequencing. PhyloID is run in two steps, each being a self-contained automated process amenable to GMP validation. The first module, MiLY, is responsible for assembling individual sequence reads into contigs, and annotating all sequences with a unique sequence identifier, the number of reads in each contig, and the length of the sequence. The trimmed, assembled and annotated data are then processed by PhyloID's second module, NGmapper. NGmapper takes the FASTA-formatted output from MiLY and identifies the taxonomic origins of the contigs and singletons therein. It compares each sequence's BLASTN hit profile against the patterns of evolutionary relationships described within phylogenomic distance matrices for all of the various taxonomic groups, in order to find the best fit. NGmapper then produces lists of taxonomic assignments in both summarized and detailed form, and tree files for viewing results graphically. We optimized PhyloID's parameters and measured its performance using simulated metagenomic data and subsets of the reference phylogenies. PhyloID's precision and recall in identifying simulated sequences were measured by information retrieval analysis, focusing on read length, read number, sequence accuracy, background complexity, taxonomy and reference data coverage. We found PhyloID to be highly accurate and quantitative in its taxonomic mapping of sequences, with excellent precision, sensitivity and robustness. The degree of taxonomic representation available in publicly available databases remains an issue, as expected, for any sequence classifier, but community sequencing efforts are poised to overcome this problem. In order to illustrate real-world usage of the application, we also describe some simple spike-recovery experiments as well as a multi-site comparative characterization of a viral suspension. These data help to illustrate, to corroborate, and to extend results using simulated data. LAY ABSTRACT: In order to address gaps in the detection of contaminating viruses and microorganisms in vaccines and other biologicals, manufacturers are exploring the use of new technologies that promise greater sensitivity and breadth of coverage. One challenge in implementing such new methods is the complexity of analysis of the "big data" generated by these new instruments: hundreds of millions of sequence reads (segments of genetic material from viruses and cells) need to be compared against a vast and growing number of entries in genetic databases, in order to come up with a confident identification. These large-scale analyses must furthermore be carried out within the strict regulatory environment that governs the industry. We have developed an automated software pipeline named PhyloID™ that is capable of identifying viruses and microorganisms from large-scale sequence data. Using simulated data as well as real samples, we show that PhyloID is both sensitive and accurate in identifying any type of potential contaminant. Such a powerful new assay will be an important addition to the adventitious agent testing package, providing further assurance about product safety.

Preliminary Evaluation of Next-Generation Sequencing Performance Relative to qPCR and In Vitro Cell Culture Tests for Human Cytomegalovirus.

To compare the performances of conventional in vitro indicator cell culture, quantitative polymerase chain reaction (qPCR), and next-generation sequencing (NGS) as detection methods for adventitious agents, a preliminary assessment was performed using human cytomegalovirus (HCMV) as a model virus. HCMV was spiked into a crude viral harvest at various concentrations and inoculated onto MRC-5 cell monolayers. The cultures were observed for cytopathic effects (CPEs) as per the compendial method requirements, and samples were taken at various time points for analysis by qPCR or NGS. When using NGS, the detection of HCMV is 10 fold more sensitive than observed using the conventional CPE endpoint method. It may be possible for qPCR to achieve the detection level demonstrated by NGS, but further optimization of the technique would be required. In addition, NGS was able to detect HCMV in the initial inoculum when it was spiked in at 10 CCID50/mL, suggesting the potential to eliminate cell culture amplification with an NGS-based assay. This study highlights the advantage of NGS as a surrogate broad-spectrum technology for the detection of adventitious agents in biologics. LAY ABSTRACT: Human cytomeglovirus (HCMV) is highly prevalent in the general population and can lead to serious health issues in both immumocompromised individuals and/or newborns. The testing of HCMV in biological materials is stipulated by multiple regulatory agencies where HCMV is a potential risk. This test involves inoculating cell lines that are susceptible to HCMV infection, incubating the cultures for 28 days, and observing the cells for signs of viral infection. Next-generation sequencing and quantitative polymerase chain reaction (qPCR) are two technologies that can potentially shorten the extended 28 day cell culture incubation. In this study, we compared the sensitivity of the compendial cell culture assay with NGS and qPCR for the detection of HCMV. Our results show that NGS can potentially achieve a detection limit that is 10 times more sensitive than the cell culture assay. In addition, NGS was able to detect HCMV in the initial inoculum, potentially eliminating the need for cell culture amplification of the virus. Finally, sequence data generated by NGS directly demonstrate the presence of HCMV, and such information can serve as the foundation for any follow-up investigation.

Determination of HSV-1 UL5 and UL29 gene copy numbers in an HSV complementing Vero cell line.

The genetic stability of transgenes is a critical characteristic used to assess constructed cell lines used for vaccine production. The evaluation of gene copy numbers by a qPCR method, is one of the most common approaches used to assess the consistency of transgenes in a constructed cell line. The cell line AV529-19 is a Vero-based cell line specifically engineered to express the HSV-1 UL5 and UL29 open reading frames. AV529-19 is used to support the replication of a defective HSV-2 viral candidate vaccine called HSV529. To assess the genetic stability of the UL5 and UL29 transgenes in AV529-19 cells, a digital PCR-based approach was developed. During characterization of the test method, the specificity, accuracy, and intermediate precision of the assay was investigated based on regulatory guidelines. The developed assay was used to monitor the stability of the transgenes in the manufactured AV529-19 cell lines by comparison of transgene copy numbers in the master cell bank (MCB) with their copy numbers in the extended cell bank (ECB). Results showed that the UL29 and UL5 transgenes are stable in that there are one and three copies of the UL29 and UL5 genes, respectively, per cell in both the AV529-19 MCB and ECB.

Short communication: evaluating the level of expressed HIV type 1 gp120 and gag proteins in the vCP1521 vector by two immunoplaque methods.

Over the past few years, several recombinant ALVAC constructs have been used as delivery systems in various vaccine research studies and trials. The ALVAC-HIV vCP1521 vector has been used as a vaccine delivery system in the RV144 study, a phase III HIV study that displayed over 31% protective efficacy. One of the important parameters for evaluating the potency of an ALVAC construct is the stable expression of proteins encoded by the inserted genes. Herein, the expression of inserted gp120 and gag genes in two manufactured ALVAC-HIV vCP1521 lots have been determined by two immunoplaque methods (dish and plaque lift). Both methods were specific and robust and demonstrated that the ALVAC-HIV vCP1521 lots were able to express gp120 and gag proteins in over 99% of the infectious plaques.