Vaccine Antigen Content Research Articles

Development and verification of an enzyme-linked immunosorbent assay for the quantification of toxoid A and toxoid B from Clostridioides difficile

Clostridioides difficile (C. difficile) is the most common cause of nosocomial antibiotic associated diarrhoea. The incidence of C. difficile infection (CDI) has been rising worldwide over the last 20 years with consequent rises in morbidity, mortality and healthcare costs, although the incidence has fallen in the UK over the last few years. Confirmation of diagnosis and early intervention are critical to the management of CDI. The standard treatment for CDI is the administration of antibiotics. However, vaccination has been recognized as the most cost-effective treatment for the prevention and possible long-term protection against CDI episode. There are several promising vaccine candidates in various stages of development. Many of these vaccines have displayed good efficacy for CDI under laboratory conditions or in clinical trials. With the emergence of vaccines against C. difficile, here we describe the development and verification of an Enzyme Linked Immunosorbent Assay (ELISA) that can be used for the quality control testing of candidate vaccines against C. difficile through the measurement of vaccine antigen content. Verification of the assay was performed by assessment of specificity, sensitivity, intermediate precision and relative accuracy. The ELISAs were specific for the toxoids being detected and the detection limit of the assay for toxoid A was 4.88 ng/mL and 3.91 ng/mL for toxoid B. The geometric coefficients of variation for intermediate precision did not exceed 25% and relative accuracy was within 77–130%. We therefore conclude that the ELISA described here is sufficiently sensitive, specific, precise and accurate for use for the quality control testing of candidate C. difficile vaccines.

Interrogation of a live-attenuated enterotoxigenic Escherichia coli vaccine highlights features unique to wild-type infection

Enterotoxigenic Escherichia coli (ETEC) infections are a common cause of severe diarrheal illness in low- and middle-income countries. The live-attenuated ACE527 ETEC vaccine, adjuvanted with double mutant heat-labile toxin (dmLT), affords clear but partial protection against ETEC challenge in human volunteers. Comparatively, initial wild-type ETEC challenge completely protects against severe diarrhea on homologous re-challenge. To investigate determinants of protection, vaccine antigen content was compared to wild-type ETEC, and proteome microarrays were used to assess immune responses following vaccination and ETEC challenge. Although molecular interrogation of the vaccine confirmed expression of targeted canonical antigens, relative to wild-type ETEC, vaccine strains were deficient in production of flagellar antigens, immotile, and lacked production of the EtpA adhesin. Similarly, vaccination ± dmLT elicited responses to targeted canonical antigens, but relative to wild-type challenge, vaccine responses to some potentially protective non-canonical antigens including EtpA and the YghJ metalloprotease were diminished or absent. These studies highlight important differences in vaccine and wild-type ETEC antigen content and call attention to distinct immunologic signatures that could inform investigation of correlates of protection, and guide vaccine antigen selection for these pathogens of global importance.

SPRi-based hemagglutinin quantitative assay for influenza vaccine production monitoring

Influenza vaccine manufacturers lack tools, whatever the involved production bioprocess (egg or cell-based), to precisely and accurately evaluate vaccine antigen content from samples. Indeed, the gold standard single-radial immunodiffusion (SRID) assay, which remains the only validated assay for the evaluation of influenza vaccine potency, is criticized by the scientific community and regulatory agencies since a decade for its high variability, lack of flexibility and low sensitivity. We hereby report an imaging surface plasmon resonance (SPRi) assay for the quantification of both inactivated vaccine influenza antigens and viral particles derived from egg- and cell-based production samples, respectively. The assay, based on fetuin-hemagglutinin interactions, presents higher reproducibility (<3%) and a greater analytical range (0.03–20 µg/mL) than SRID for bulk monovalent and trivalent vaccine and its limit of detection was evaluated to be 100 times lower than the SRID’s one. Finally, viral particles production through cell culture-based bioprocess was also successfully monitored using our SPRi-based assay and a clear correlation was found between the biosensor response and total virus particle content.

Establishing the 1st Chinese National Standard for inactivated hepatitis A vaccine

A reference standard calibrated in the International Units is needed for the quality control of hepatitis A vaccine. Thus, National Institutes for Food and Drug Control launched a project to establish a non-adsorbed inactivated hepatitis A vaccine reference as the working standard calibrated against the 1st International Standard (IS). Two national standard candidates (NSCs) were obtained from two manufacturers, and designated as NSC A (lyophilized form) and NSC B (liquid form). Six laboratories participated in the collaborative study and were asked to use their in-house validated enzyme-linked immunosorbent assay methods to detect hepatitis A vaccine antigen content. Although both exhibited good parallelism and linear relationship with IS, NSC B showed a better agreement among laboratories than NSC A. And based on suitability of the candidates, NSC B was selected. The accelerated degradation study showed that NSC B was stable at the storage temperature (≤−70 °C). Therefore NSC B was approved as the first Chinese national antigen standard for inactivated hepatitis A vaccine, with an assigned antigen content of 70 IU/ml.

Molecular Epidemiology of Group A Streptococcus Infections in Cambodian Children, 2007-2012.

To the Editors: Group A Streptococcus (GAS) is responsible for significant morbidity and mortality globally. Two strong vaccine candidates are currently under evaluation: a 30-valent type–specific M protein–based vaccine and a vaccine targeting the conserved J8 region of M protein.1 The 30-valent vaccine covers the most frequent serotypes circulating in high-income countries, but coverage may be suboptimal in low-income settings with greater GAS emm-type diversity.2 Sixty-eight allelic variants have been described for J8, and the relation between allelic diversity and vaccine efficacy is unclear.1 Limited epidemiologic data are available from many regions making vaccine coverage estimates imprecise. There are no previous data for low-income countries in South East Asia.2 The clinical microbiology database at Angkor Hospital for Children, a nongovernmental pediatric hospital serving the population of northern Cambodia, was searched to identify clinical GAS isolates cultured between January 1, 2007 and December 31, 2012. These isolates underwent molecular emm-typing. emm-clusters, and the J8 vaccine antigen content, were deduced from the emm-typing result as previously described.3 The 30-valent vaccine coverage was estimated using currently available cross-opsonization data.3,4 Strain diversity was assessed by Simpson Reciprocal Index. One hundred fifty GAS isolates from 149 patients were characterized. The median patient age was 3.8 years (range: 0–18.6). One hundred eighteen (78.6%) isolates were from skin and soft tissue infections, 16 (10.7%) from bloodstream infections, 7 (4.7%) from bone/joint infections, 7 (4.7%) from pharyngitis and 2 (1.3%) from infections at other sites. Fifty emm-types were identified from 13 emm-clusters and 2 isolates were considered nontypeable (see Table, Supplemental Digital Content, https://links.lww.com/INF/C234). No novel emm-types were identified. The Simpson Reciprocal Index was 28.5 (95% confidence interval: 23.1–37.3) indicating considerable diversity, similar to that seen in other low-income countries.2 Potential coverage of the J8 vaccine was predicted to be excellent with 43 (28.7%) and 104 (69.3%) isolates predicted to have the J8 and J8.1 allele, respectively. Therefore, J8 vaccine coverage could be expected to be 98.0% (95% confidence interval: 94.2%–99.6%). Fifty isolates (33.3%) were of emm-types covered by the 30-valent vaccine, and an additional 42 isolates (28.0%) have been shown to be potentially covered by the vaccine as a result of cross-opsonization. Therefore, the potential coverage could be expected to be 61.3% (95% confidence interval: 53.0%–69.2%) but may be higher because 26.0% of the isolates belong to 16 emm-types, which have not yet been examined for evidence of cross-opsonization. Comparison with the only other available regional data set revealed considerably greater emm-type diversity in the Cambodian isolates compared with those isolated in Thailand between 1985 and 2004.5 Fifty-nine emm-types from 13 emm-clusters were represented in the combined data set. Only 8 of the 13 emm-clusters were found in Thailand. There were 10 shared emm-types, comprising 64.2% of the Thailand isolates but only 26.4% of the Cambodia isolates. Our study had several limitations. Pharyngeal isolates were not well represented because of clinician sampling practices and also there was an absence of adult sampling in the data set. Also, the population studied may not be representative of Cambodia as a whole. Finally, comparisons between the current data and the Thailand data set are limited by the differences between the study populations and nonoverlapping study periods. Overall, these data indicate a high diversity of circulating GAS strains in Cambodia, the potential high coverage of the J8 vaccine candidate and the need for complementary studies to assess the potential coverage of the 30-valent vaccine candidate. These results highlight the need for robust regional and country-level data for vaccine planning purposes. Paul Turner, MB BS, PhD, FRCPCH, FRCPath Cambodia-Oxford Medical Research Unit Angkor Hospital for Children, Siem Reap, Cambodia, Centre for Tropical Medicine and Global Health Nuffield Department of Medicine University of Oxford Oxford, United Kingdom Pises Ngeth, MD Angkor Hospital for Children Siem Reap, Cambodia Claudia Turner, MB BS, PhD, FRCPCH Cambodia-Oxford Medical Research Unit Angkor Hospital for Children, Siem Reap, Cambodia Centre for Tropical Medicine and Global Health Nuffield Department of Medicine University of Oxford Oxford, United Kingdom Sena Sao Angkor Hospital for Children Siem Reap, Cambodia Nicholas P. J. Day, MD, FRCP Centre for Tropical Medicine and Global Health Nuffield Department of Medicine University of Oxford Oxford, United Kingdom Mahidol-Oxford Tropical Medicine Research Unit Faculty of Tropical Medicine Mahidol University Bangkok, Thailand Ciara Baker, MSc Group A Streptococcal Research Group Murdoch Childrens Research Institute Melbourne, Australia Andrew C. Steer, MB BS, PhD, FRACP Pierre R. Smeesters, MD, PhD Group A Streptococcal Research Group Murdoch Childrens Research Institute Centre for International Child Health University of Melbourne Melbourne, Australia

Assessment of the immunogenicity and safety of varying doses of an MF59®-adjuvanted cell culture-derived A/H1N1 pandemic influenza vaccine in Japanese paediatric, adult and elderly subjects

IntroductionEffective vaccination strategies are required to combat future influenza pandemics. Here we report the results of three independent clinical trials performed in Japan to assess the immunogenicity, tolerability and safety of varying doses of a cell culture-derived MF59®-adjuvanted A/H1N1 pandemic vaccine in healthy Japanese paediatric, adult and elderly subjects. MethodsOne hundred and twenty-three children (6 months–18 years), and 200 adults (19–60 years) were randomly assigned in a 1:1 ratio to receive two doses of vaccine containing either 7.5μg antigen with a full (9.75mg) adjuvant dose, or 3.75μg antigen with a half (4.875mg) adjuvant dose. One hundred elderly (≥61 years) subjects received only the low antigen/adjuvant vaccine formulation. Immunogenicity was assessed by haemagglutination inhibition assay at baseline and three weeks after the first and second vaccine doses on Days 22 and 43, respectively. Solicited and unsolicited adverse reactions were recorded for seven and 21 days post-immunization, respectively. ResultsIn adult and elderly subjects, a single low antigen/adjuvant dose vaccination was sufficient to meet all of the three European licensure criteria established for influenza vaccines. One high, or two low antigen/adjuvant dose vaccinations were required to meet the licensure criteria in paediatric subjects. Both vaccine formulations were well tolerated, with the majority of adverse reactions mild to moderate in severity. None of the five serious adverse events reported throughout the three trials were considered to be vaccine-related by the investigators. ConclusionThe use of MF59 adjuvant allows for much reduced vaccine antigen content, and a single dose administration schedule in adults and the elderly. The production of pandemic vaccine using modern cell culture techniques is highly advantageous in terms of the quantity, quality, and rapidity of antigen production; these benefits, in combination with the use of MF59, maximize manufacturing capacity and global vaccine supply. These data support the suitability of the investigational vaccine for use in the Japanese paediatric, adult, and elderly populations.

IgG responses after booster vaccination with different pertussis vaccines in Dutch children 4 years of age: Effect of vaccine antigen content

Since whooping cough is reemerging in the Netherlands from 1996 onwards, several changes in the national immunization program have been implemented regarding the pertussis vaccinations. The aim of this study is to investigate IgG responses in whole cell (wP) and acellular (aP) pertussis vaccine primed children following revaccination with different pertussis booster vaccines at 4 years of age. IgG levels to pertussis toxin (Pt), filamentous heamagglutin (FHA), pertactin (Prn) and fimbriae type 2 and 3 (Fim2/3) and avidities of Pt and Prn antibodies were measured using a multiplex immunoassay. Before and after the booster we found significantly higher IgG levels to Pt, FHA and Prn in aP compared to wP primed children. In all children a booster vaccination with a pertussis vaccine containing a high antigen dose ( Infanrix™) induced higher IgG responses compared to a low antigen dose containing vaccine ( Triaxis™). Avidities of Pt- and Prn-antibodies before and after booster vaccination were significantly higher in aP than in wP primed children. This study shows that a booster vaccine with high pertussis antigen concentrations induces higher antibody levels than a low antigen containing vaccine. In children primed with the Dutch DTwP-IPV-Hib vaccine we suggest to administer a booster vaccine containing high pertussis antigens to optimize IgG responses. The pertussis vaccination history has to be taken into account in decisions on changes in pertussis vaccination policy.

Special Edition: Editorial

It is surprising that over 60 years have elapsed since the first use of influenza vaccines, yet with the advances in biotechnology that have occurred in the interim, the majority of the influenza vaccines that we use today are still non‐living viral antigens produced in a relatively uncontrolled substrate, the embryonated chicken egg. It is also surprising that the impact of seasonal influenza has only gradually entered the consciousness of public health authorities over the last couple of decades. This has resulted in expanded national recommendations for influenza vaccination and gradually increasing vaccine use, at least in many of the more economically developed and rapidly developing countries. 1 , 2 It has been widely accepted for many years that a future influenza pandemic is inevitable. Regardless of the development of new influenza‐specific antivirals, vaccines are seen as the most effective response against such a threat and, therefore, have a key role in pandemic preparedness planning. It is quite alarming, then, that the current global capacity to produce vaccine would fall woefully short of global needs in the event of a pandemic that occurred today. 3 , 4 One of the few significant ‘improvements’ introduced into vaccine manufacture was a move to sub‐virion vaccines in order to reduce the reactogenicity that was observed principally when administered to infants and young children. 5 Ironically, this was clearly demonstrated in the late 1970s to reduce vaccine immunogenicity in immunologically naive individuals, 6 , 7 , 8 as would be the case in a pandemic. Other studies have also shown reduced immunogenicity of seasonal sub‐virion vaccine in the older adult, a major target group for annual vaccination. 9 Another improvement, the immunological standardization of vaccine antigen content, 10 , 11 might, in itself, contribute to delays in vaccine availability due to the time required to produce the required reagents. The ongoing epizootic of avian influenza due to A(H5N1) viruses, and the growing count of associated human fatalities, has stimulated the development of candidate pandemic vaccines with an emphasis on the H5 subtype. 12 All of the initial human studies with H5 vaccines suggested that the haemagglutinin of this subtype displayed an immunogenicity that was lower than that of the haemagglutinins of other candidate pandemic viruses, such as H2 and H9 13 , 14 , 15 which has compounded the shortcomings of the current sub‐virion vaccines and further reduces the potential pandemic vaccine supply. This has stimulated a variety of approaches to resolving this issue including new ways of producing vaccine antigen, improving immunogenicity by the use of existing and new immunological adjuvants and even a return to the use of whole virus preparations. A number of encouraging results that offer the possibility of improving the quantity and potency of A(H5N1) vaccines within the immediate future have recently been reported. In view of their importance to those involved in public health programmes and pandemic preparedness planning it is important to record as many of these as possible in a single publication. We have, therefore, canvassed widely among vaccine manufacturers and developers for up‐to‐date review articles describing their improved products for seasonal and/or pandemic influenza. Although not all have contributed there has been an excellent response and the journal is pleased to publish these articles in this special edition.

Quantification of infectious bursal disease viral proteins 2 and 3 in inactivated vaccines as an indicator of serological response and measure of potency

Viral protein 2 and viral protein 3 (VP2 and VP3) were quantified in a series of inactivated infectious bursal disease oil emulsion vaccines using enzyme-linked immunosorbent assay, and the dependence of the serological response on vaccine antigen content was studied. Large differences in antigen content, up to 50-fold, were found between vaccines. Neutralizing antibody titres at 3 to 6 weeks after vaccination varied from 3 log2 to 16 log2. None of the vaccines induced an antibody titre equal to that of the reference serum used as an indicator of sufficient potency in the European Pharmacopoeia. Neutralizing antibody titres after vaccination correlated highly with the VP2 content of the vaccines. A significant correlation was also found between the VP3 content and the antibody response. Our data illustrate that the antigen content of inactivated infectious bursal disease vaccines is a reliable indicator of the protective serological response after vaccination, and consequently could be used as a measure of vaccine potency. This holds true for both VP2, the antigen that induces neutralizing antibodies, as well as for VP3, which does not induce neutralizing antibodies.

A collaborative study on the use of single radial immunodiffusion for the assay of rabies virus glycoprotein

The single radial immunodiffusion (SRD) technique has been applied to the assay of the glycoprotein content of rabies vaccines produced in cell cultures. Fourteen laboratories in seven countries participated in a collaborative study to evaluate the reproducibility of the SRD technique; some laboratories also examined vaccines in the mouse protection (NIH) test and by enzyme immunoassay. Good agreement was found between potency estimates using the SRD technique: the geometric coefficients of variation for combined potency estimates of all laboratories were about 10%. SRD assays appear to have a role for the in vitro assay of antigen content of vaccine and could complement results obtained in in vivo assays which are subject to wide variability.