Vaccine Lots Research Articles

Adaptive immune response to whole cell pertussis vaccine reflects vaccine quality: A possible complementation to the Pertussis Serological Potency test

Whole cell Bordetella pertussis (wP) vaccines are still used in many countries to protect against the respiratory disease pertussis. The potency of whole-cell pertussis vaccine lots is determined by an intracerebral challenge test (the Kendrick test). This test is criticized due to lack of immunological relevance of the read-out after an intracerebral challenge with B. pertussis. The alternative in vivo test, which assesses specific antibody levels in serum after wP vaccination, is the Pertussis Serological Potency test (PSPT). Although the PSPT focuses on a parameter that contributes to protection, the protective immune mechanisms after wP vaccination includes more elements than specific antibody responses only. In this study, additional parameters were investigated, i.e. circulating pro-inflammatory cytokines, antibody specificity and T helper cell responses and it was evaluated whether they can be used as complementary readout parameters in the PSPT to assess wP lot quality. By deliberate manipulation of the vaccine preparation procedure, a panel of high, intermediate and low quality wP vaccines were made. The results revealed that these vaccines induced similar IL-6 and IP10 levels in serum 4h after vaccination (innate responses) and similar antibody levels directed against the entire bacterium. In contrast, the induced antibody specificity to distinct wP antigens differed after vaccination with high, intermediate and low quality wP vaccines. In addition, the magnitude of wP-induced Th cell responses (Th17, Th1 and Th2) was reduced after vaccination with a wP vaccine of low quality. T cell responses and antibody specificity are therefore correlates of qualitative differences in the investigated vaccines, while the current parameter of the PSPT alone was not sensitive enough to distinguish between vaccines of different qualities. This study demonstrates that assessment of the magnitude of Th cell responses and the antigen specificity of antibodies induced by wP vaccination could form valuable complementary parameters to the PSPT.

Safety, immune lot-to-lot consistency and non-inferiority of a fully liquid pentavalent DTwP-HepB-Hib vaccine: Results from Phase III licensure study of Shan5™

Background: Pentavalent combination vaccines perform a key role in increasing vaccine coverage rate; and provide an efficient and reliable method of implementing WHO recommendations for controlling diphtheria, tetanus, pertussis, hepatitis B and Hib infections on a worldwide basis. Methods & Materials: Study design: A phase III, multi-center, randomized, single blinded study of a fully liquid pentavalent DTwP-HepB-Hib investigational vaccine (Shan5™) was conducted across India in healthy toddlers and infants. Cohort 1 consisted of 15 toddlers aged 15-18 months, administered with a single booster dose; Cohort 2 consisted of 1085 infants aged 6-8 weeks, administered 3 doses at 6-8, 10-12 and 14-16 weeks of age. Objectives: Subjects in Cohort 1 were evaluated for safety and immunogenicity. Immunogenicity and safety were evaluated in Cohort 2 subjects vaccinated with a three-dose primary immunization of either the investigational vaccine or a locally licensed comparator vaccine (Pentavac™ SD). Immune consistency analysis among three lots of the investigational vaccine, and immune non-inferiority analysis of pooled (three lots) data of investigational vaccine versus comparator vaccine were also evaluated in cohort 2. Results: The vaccines demonstrated comparable safety and immune responses in cohort 1. In cohort 2, immune non-inferiority against the comparator vaccine (primary endpoint) was demonstrated for all five antigens and safety results (secondary endpoint) were comparable between vaccine groups. Equivalent immune consistency among three lots was observed for all antigens except whole cell pertussis antigens, where a marginal variation was observed. The variation was linked to the low power of the test and concluded to be clinically insignificant. Conclusion: The investigational, fully-liquid, whole-cell pertussis (wP) containing pentavalent vaccine has a good safety profile and immunologically non-inferior to the licensed comparator vaccine. This study was the basis for licensure in India and also WHO- prequalification for international use.

Effect of different detoxification procedures on the residual pertussis toxin activities in vaccines

Pertussis toxin (PTx) is a major virulence factor produced by Bordetella pertussis and its detoxified form is one of the major protective antigens in vaccines against whooping cough. Ideally, PTx in the vaccine should be completely detoxified while still preserving immunogenicity. However, this may not always be the case. Due to multilevel reaction mechanisms of chemical detoxification that act on different molecular sites and with different production processes, it is difficult to define a molecular characteristic of a pertussis toxoid. PTx has two functional distinctive domains: the ADP-ribosyltransferase enzymatic subunit S1 (A-protomer) and the host cell binding carbohydrate-binding subunits S2–5 (B-oligomer); and in this study, we investigated the effect of different detoxification processes on these two functional activities of the residual PTx in toxoids and vaccines currently marketed worldwide using a recently developed in vitro biochemical assay system. The patho-physiological activities in these samples were also estimated using the in vivo official histamine sensitisation tests. Different types of vaccines, detoxified by formaldehyde, glutaraldehyde or by both, have different residual functional and individual baseline activities. Of the vaccines tested, PT toxoid detoxified by formaldehyde had the lowest residual PTx ADP-ribosyltransferase activity. The carbohydrate binding results detected by anti-PTx polyclonal (pAb) and anti-PTx subunits monoclonal antibodies (mAb) showed specific binding profiles for toxoids and vaccines produced from different detoxification methods. In addition, we also demonstrated that using pAb or mAb S2/3 as detection antibodies would give a better differential difference between these vaccine lots than using mAbs S1 or S4. In summary, we showed for the first time that by measuring the activities of the two functional domains of PTx, we could characterise pertussis toxoids prepared from different chemical detoxification methods and this study also highlights the potential use of this in vitro biochemical assay system for in-process control.

Eliminating Vaccine- Preventable Diseases Around The World.

To ensure immunization access, equity, and sustainability, vaccines must remain a top priority for governments across the globe.

A High-Throughput Size Exclusion Chromatography Method to Determine the Molecular Size Distribution of Meningococcal Polysaccharide Vaccine.

Molecular size distribution of meningococcal polysaccharide vaccine is a readily identifiable parameter that directly correlates with the immunogenicity. In this paper, we report a size exclusion chromatography method to determine the molecular size distribution and distribution coefficient value of meningococcal polysaccharide serogroups A, C, W, and Y in meningococcal polysaccharide (ACWY) vaccines. The analyses were performed on a XK16/70 column packed with sepharose CL-4B with six different batches of Ingovax® ACWY, a meningococcal polysaccharide vaccine produced by Incepta Vaccine Ltd., Bangladesh. A quantitative rocket immunoelectrophoresis assay was employed to determine the polysaccharide contents of each serogroup. The calculated distribution coefficient values of serogroups A, C, W, and Y were found to be 0.26 ± 0.16, 0.21 ± 0.11, 0.21 ± 0.11, and 0.14 ± 0.12, respectively, and met the requirements of British Pharmacopeia. The method was proved to be robust for determining the distribution coefficient values which is an obligatory requirement for vaccine lot release.

Deep sequencing analysis of the heterogeneity of seed and commercial lots of the bacillus Calmette-Guérin (BCG) tuberculosis vaccine substrain Tokyo-172.

BCG, only vaccine available to prevent tuberculosis, was established in the early 20th century by prolonged passaging of a virulent clinical strain of Mycobacterium bovis. BCG Tokyo-172, originally distributed within Japan in 1924, is one of the currently used reference substrains for the vaccine. Recently, this substrain was reported to contain two spontaneously arising, heterogeneous subpopulations (Types I and II). The proportions of the subpopulations changed over time in both distributed seed lots and commercial lots. To maintain the homogeneity of live vaccines, such variations and subpopulational mutations in lots should be restrained and monitored. We incorporated deep sequencing techniques to validate such heterogeneity in lots of the BCG Tokyo-172 substrain without cloning. By bioinformatics analysis, we not only detected the two subpopulations but also detected two intrinsic variations within these populations. The intrinsic variants could be isolated from respective lots as colonies cultured on plate media, suggesting analyses incorporating deep sequencing techniques are powerful, valid tools to detect mutations in live bacterial vaccine lots. Our data showed that spontaneous mutations in BCG vaccines could be easily monitored by deep sequencing without direct isolation of variants, revealing the complex heterogeneity of BCG Tokyo-172 and its daughter lots currently in use.

Safety, immune lot-to-lot consistency and non-inferiority of a fully liquid pentavalent DTwp-HepB-Hib vaccine in healthy Indian toddlers and infants

ABSTRACTPentavalent combination vaccines are important tools to strengthen the immunization programs in numerous countries throughout the world. A large number of countries have recognized the value of combination vaccines and have introduced whole cell pentavalent vaccines into their immunization programs. A phase III, multi-center, randomized, single blinded study of a fully liquid pentavalent DTwP-HepB-Hib investigational vaccine (Shan5™) was conducted across India in 2 cohorts: 15 toddlers were evaluated for safety and immunogenicity following a single booster dose (Cohort 1) followed by 1085 infants (Cohort 2) evaluated for immunogenicity and safety following 3-dose primary immunization of the investigational vaccine or a locally licensed comparator vaccine (Pentavac SD). Immune consistency analysis among 3 lots of the investigational vaccine, and immune non-inferiority analysis of pooled (3 lots) data of investigational vaccine vs. comparator vaccine were carried out in cohort 2. The vaccines demonstrated comparable safety and immune responses in cohort 1. In cohort 2, equivalent immune consistency among 3 lots was observed for all antigens except whole cell pertussis antigens, where a marginal variation was observed which was linked to the low power of the test and concluded to not have any clinical significance. Immune non-inferiority against the comparator vaccine was demonstrated for all 5 antigens. Safety results were comparable between vaccine groups. This investigational, fully-liquid, whole-cell pertussis (wP) containing new pentavalent vaccine was found to be safe and immunologically non-inferior to the licensed comparator vaccine.

Study of rabies virus by Differential Scanning Calorimetry

Differential Scanning Calorimetry (DSC) has been used in the past to study the thermal unfolding of many different viruses. Here we present the first DSC analysis of rabies virus. We show that non-inactivated, purified rabies virus unfolds cooperatively in two events centered at approximately 62 and 73°C. Beta-propiolactone (BPL) treatment does not alter significantly viral unfolding behavior, indicating that viral inactivation does not alter protein structure significantly. The first unfolding event was absent in bromelain treated samples, causing an elimination of the G-protein ectodomain, suggesting that this event corresponds to G-protein unfolding. This hypothesis was confirmed by the observation that this first event was shifted to higher temperatures in the presence of three monoclonal, G-protein specific antibodies. We show that dithiothreitol treatment of the virus abolishes the first unfolding event, indicating that the reduction of G-protein disulfide bonds causes dramatic alterations to protein structure. Inactivated virus samples heated up to 70°C also showed abolished recognition of conformational G-protein specific antibodies by Surface Plasmon Resonance analysis. The sharpness of unfolding transitions and the low standard deviations of the Tm values as derived from multiple analysis offers the possibility of using this analytical tool for efficient monitoring of the vaccine production process and lot to lot consistency.

Genetic stability of live attenuated vaccines against potentially pandemic influenza viruses

BackgroundEnsuring genetic stability is a prerequisite for live attenuated influenza vaccine (LAIV). This study describes the results of virus shedding and clinical isolates’ testing of Phase I clinical trials of Russian LAIVs against potentially pandemic influenza viruses in healthy adults. MethodsThree live attenuated vaccines against potentially pandemic influenza viruses, H2N2 LAIV, H5N2 LAIV and H7N3 LAIV, generated by classical reassortment in eggs, were studied. For each vaccine tested, subjects were randomly distributed into two groups to receive two doses of either LAIV or placebo at a 3:1 vaccine/placebo ratio. Nasal swabs were examined for vaccine virus shedding by culturing in eggs and by PCR. Vaccine isolates were tested for temperature sensitivity and cold-adaptation (ts/ca phenotypes) and for nucleotide sequence. ResultsThe majority of nasal wash positive specimens were detected on the first day following vaccination. PCR method demonstrated higher sensitivity than routine virus isolation in eggs. None of the placebo recipients had detectable vaccine virus replication.All viruses isolated from the immunized subjects retained the ts/ca phenotypic characteristics of the master donor virus (MDV) and were shown to preserve all attenuating mutations described for the MDV. These data suggest high level of vaccine virus genetic stability after replication in humans.During manufacture process, no additional mutations occurred in the genome of H2N2 LAIV. In contrast, one amino acid change in the HA of H7N3 LAIV and two additional mutations in the HA of H5N2 LAIV manufactured vaccine lot were detected, however, they did not affect their ts/ca phenotypes. ConclusionsOur clinical trials revealed phenotypic and genetic stability of the LAIV viruses recovered from the immunized volunteers. In addition, no vaccine virus was detected in the placebo groups indicating the lack of person-to-person transmission.LAIV TRIAL REGISTRATION at ClinicalTrials.gov: H7N3-NCT01511419; H5N2-NCT01719783; H2N2-NCT01982331.

Standardization of the methods and reference materials used to assess virus content in varicella vaccines.

BackgroundIn Korea, every vaccine lot is tested by the National Center for Lot Release (NCLR) in accordance with the national lot release procedures to ensure the safety and efficacy of vaccines. These quality tests examine the virus content in varicella vaccines via plaque assays (either the agar overlay method [AOM] or plaque staining method [PSM]), according to the procedures suggested by the Korean Reference Material for the Varicella Vaccine (KRMVV) or the manufacturer’s standard in-house protocol.AimTo standardize the virus content tests, viral titers in the KRMVV were measured using the PSM at four participating laboratories in a collaborative study. With the aim of developing a standardized method using the KRMVV as a positive control, we compared the ability of the two test methods, AOM and PSM, to accurately and reproducibly determine the virus content of two commercial varicella vaccines.ResultsThe results showed that the standardized method (PSM) was more suitable for quality control analysis of the varicella vaccine.ConclusionUse of a standardized method (PSM) according to the Korean reference material will improve the reliability and objectivity of lot release testing.

Phase III, randomized controlled trial in girls 9-15 years old to evaluate lot consistency of a novel nine-valent human papillomavirus L1 virus-like particle vaccine

A 9-valent human papillomavirus (6/11/16/18/31/33/45/52/58) VLP (9vHPV) vaccine has recently been proven highly efficacious in preventing disease associated with vaccine HPV types in a pivotal Phase III study. The demonstration of lot-to-lot consistency to confirm the reliability of the manufacturing process is a regulatory requirement for vaccine licensure in the United States. A randomized trial was conducted to demonstrate that three lots of 9vHPV vaccine elicit equivalent antibody response for all 9 vaccine types. The study required thorough planning because it required success on 27 separate statistical comparisons. An innovative statistical approach was used taking into account between-lot variance for more conservative power calculations. The study demonstrated equivalence of three lots of 9vHPV vaccine for all 9 vaccine types.

Analysis of the genetic stability of the master virus seeds for live attenuated influenza vaccine production

Objective To analyze the genetic stability of master virus seed lots of live attenuated influenza vaccine A/17/California/2009/38(H1N1) and A/17/Perth/09/87(H3N2) strains. Methods The master virus seed lots were inoculated into chicken eggs for subculture. The complete genome of the 2nd, 3rd, 5th and 10th generations of viruses were amplified and sequenced. The genes encoding hemagglutinin (HA) and neuraminidase (NA) were compared with those of the WHO recommended circulating wild-type virus strains used for vaccine production in northern hemisphere during 2011-2012 influenza season. Six internal genes (PB2, PB1, PA, NP, M and NS) of each virus generation were compared with their master donor virus strain (A/Leningrad/134/17/57) for the evaluation of the genetic stability. Results The mutation rates of H1N1 and H3N2 strains after 10 passages were 0.035% and 0.022%, respectively. No mutations were found at the critical sites for controling thecold adapted (ca), temperature sensitive (ts) and attenuated (att) phenotypes. Conclusion The live attenuated influenza vaccine strains possessed high genetic stability as their tenth generations still shared 99% of homology with the original seed lots. All of the working virus seed lots met the requirements of Pharmacopoeia of the People′s Republic of China (2010 edition). Key words: Live attenuated influenza virus vaccine; Genetic stability; PCR

Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors.

The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10(24)-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10(68)-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches.

Genetic Determinants of Japanese Encephalitis Virus Vaccine Strain SA14-14-2 That Govern Attenuation of Virulence in Mice.

The safety and efficacy of the live-attenuated Japanese encephalitis virus (JEV) SA14-14-2 vaccine are attributed to mutations that accumulated in the viral genome during its derivation. However, little is known about the contribution that is made by most of these mutations to virulence attenuation and vaccine immunogenicity. Here, we generated recombinant JEV (rJEV) strains containing JEV SA14-14-2 vaccine-specific mutations that are located in the untranslated regions (UTRs) and seven protein genes or are introduced from PCR-amplified regions of the JEV SA14-14-2 genome. The resulting mutant viruses were evaluated in tissue culture and in mice. The authentic JEV SA14-14-2 (E) protein, with amino acid substitutions L107F, E138K, I176V, T177A, E244G, Q264H, K279M, A315V, S366A, and K439R relative to the wild-type rJEV clone, was essential and sufficient for complete attenuation of neurovirulence. Individually, the nucleotide substitution T39A in the 5' UTR (5'-UTR-T39A), the capsid (C) protein amino acid substitution L66S (C-L66S), and the complete NS1/2A genome region containing 10 mutations each significantly reduced virus neuroinvasion but not neurovirulence. The levels of peripheral virulence attenuation imposed by the 5'-UTR-T39A and C-L66S mutations, individually, were somewhat mitigated in combination with other vaccine strain-specific mutations, which might be compensatory, and together did not affect immunogenicity. However, a marked reduction in immunogenicity was observed with the addition of the NS1/2A and NS5 vaccine virus genome regions. These results suggest that a second-generation recombinant vaccine can be rationally engineered to maximize levels of immunogenicity without compromising safety. The live-attenuated JEV SA14-14-2 vaccine has been vital for controlling the incidence of disease caused by JEV, particularly in rural areas of Asia where it is endemic. The vaccine was developed >25 years ago by passaging wild-type JEV strain SA14 in tissue cultures and rodents, with intermittent tissue culture plaque purifications, to produce a virus clone that had adequate levels of attenuation and immunogenicity. The vaccine and parent virus sequences were later compared, and mutations were identified throughout the vaccine virus genome, but their contributions to attenuation were never fully elucidated. Here, using reverse genetics, we comprehensively defined the impact of JEV SA14-14-2 mutations on attenuation of virulence and immunogenicity in mice. These results are relevant for quality control of new lots of the current live-attenuated vaccine and provide insight for the rational design of second-generation, live-attenuated, recombinant JEV vaccine candidates.

Safety, immunogenicity, and lot-to-lot consistency of a quadrivalent inactivated influenza vaccine in children, adolescents, and adults: A randomized, controlled, phase III trial

BackgroundInactivated quadrivalent influenza vaccine (IIV4) containing two influenza A strains and one strain from each B lineage (Yamagata and Victoria) may offer broader protection against seasonal influenza than inactivated trivalent influenza vaccine (IIV3), containing a single B strain. This study examined the safety, immunogenicity, and lot consistency of an IIV4 candidate. MethodsThis phase III, randomized, controlled, multicenter trial in children/adolescents (9 through 17 years) and adults (18 through 60 years) was conducted in Australia and in the Philippines in 2012. The study was double-blind for IIV4 lots and open-label for IIV4 vs IIV3. Children/adolescents were randomized 2:2:2:1 and adults 10:10:10:1 to receive one of three lots of IIV4 or licensed IIV3. Safety data were collected for up to 6 months post-vaccination. Hemagglutination inhibition and seroneutralization antibody titers were assessed pre-vaccination and 21 days post-vaccination. Results1648 adults and 329 children/adolescents received IIV4, and 56 adults and 55 children/adolescents received IIV3. Solicited reactions, unsolicited adverse events, and serious adverse events were similar for IIV3 and IIV4 recipients in both age groups. Injection-site pain, headache, malaise, and myalgia were the most frequently reported solicited reactions, most of which were mild and resolved within 3 days. No vaccine-related serious adverse events or deaths were reported. Post-vaccination antibody responses, seroconversion rates, and seroprotection rates for the 3 strains common to both vaccines were comparable for IIV3 and IIV4 in both age groups. Antibody responses to IIV4 were equivalent among vaccine lots and comparable between age groups for each of the 4 strains. IIV4 met all European Medicines Agency immunogenicity criteria for adults for all 4 strains. ConclusionsIn both age groups, IIV4 was well tolerated and caused no safety concerns, induced robust antibody responses to all 4 influenza strains, and met all EMA immunogenicity criteria for adults. Clinical trial registry numberNCT01481454.

In vitro innate immune cell based models to assess whole cell Bordetella pertussis vaccine quality: A proof of principle

Lot release testing of vaccines is primarily based on animal models that are costly, time-consuming and sometimes of questionable relevance. In order to reduce animal use, functional in vitro assays are being explored as an alternative approach for the current lot release testing paradigm. In this study, we present an evaluation of APC platforms assessing innate immune activation by whole cell Bordetella pertussis (wP) vaccines. Primary monocytes, monocyte-derived DC (moDC) and human monocyte/DC cell lines (MonoMac6 and MUTZ-3) were compared for their capacity to respond to wP vaccines of varying quality. To produce such vaccines, the production process of wP was manipulated, resulting in wP vaccines covering a range of in vivo potencies. The responses of MUTZ-3 cells and primary monocytes to these vaccines were marginal and these models were therefore considered inappropriate. Importantly, moDC and MonoMac6 cells responded to the wP vaccines and discriminated between vaccines of varying quality, although slight variations in the responses to wP vaccines of similar quality were also observed. This study provides a proof of principle for the use of in vitro APC platforms as part of a new strategy to assess wP vaccine lot consistency, though careful standardisation of assay conditions is necessary.

Deep sequencing of the yellow fever vaccine strain 17D reveals low diversity compared to wild-type parent Asibi virus and population stability for primary and secondary seed lots of 17D vaccine

Deep sequencing of the yellow fever vaccine strain 17D reveals low diversity compared to wild-type parent Asibi virus and population stability for primary and secondary seed lots of 17D vaccine

Behind the scenes in public health: Adverse events following immunization (AEFI) signal investigation in British Columbia.

In British Columbia, vaccine safety is monitored through a passive surveillance system with voluntary reporting of adverse events following immunization (AEFIs) from immunizers to five regional health authorities and onward to the British Columbia Centre for Disease Control (BCCDC). To review and summarize all documented AEFI cluster or signal investigations carried out by BCCDC between November 2007 and July 2014. Documented cluster or signal investigations were reviewed to summarize year, alerting mechanism, event type and vaccine, investigative analysis approach, results, and public health actions. The findings and public health actions of two cluster investigations are described in detail. There were two fatality investigations and thirteen cluster investigations. The two fatalities were found to be due to sudden infant death syndrome and were not vaccine-related. Clusters were predominantly identified through notification from regional medical health officers or public health nurses, and the majority were local injection site reactions (54%), or allergic events (39%). Most investigations did not identify a specific association to a vaccine or a lot of vaccine, and no public health actions were taken. Two recent investigations-reports of hypotonic-hyporesponsive episodes with or without severe vomiting and diarrhea following receipt of a single lot of DPT-IPV/Hib/hepatitis B vaccine, and reports of severe pain past nearest joint following administration of a single lot of influenza vaccine-were thought to be vaccine-related. The former investigation did not find an association to vaccine, while the severe local reactions post-influenza immunization were determined to be a result of improper injection technique. Public health actions included communication to federal/provincial/territorial vaccine safety partners and additional injection technique training. This investigative aspect of public health immunization programs is often not in the public eye but is an important component of behind the scenes activities that serve to protect public safety.

Particle quantification of influenza viruses by high performance liquid chromatography

The influenza virus continuously undergoes antigenic evolution requiring manufacturing, validation and release of new seasonal vaccine lots to match new circulating strains. Although current production processes are well established for manufacturing seasonal inactivated influenza vaccines, significant limitations have been underlined in the case of pandemic outbreaks. The World Health Organization called for a global pandemic influenza vaccine action plan including the development of new technologies. A rapid and reliable method for the quantification of influenza total particles is crucially needed to support the development, improvement and validation of novel influenza vaccine manufacturing platforms. This work presents the development of an ion exchange-high performance liquid chromatography method for the quantification of influenza virus particles. The method was developed using sucrose cushion purified influenza viruses A and B produced in HEK 293 suspension cell cultures. The virus was eluted in 1.5M NaCl salt with 20mM Tris–HCl and 0.01% Zwittergent at pH 8.0. It was detected by native fluorescence and the total analysis time was 13.5min. A linear response range was established between 1×109 and 1×1011 virus particle per ml (VP/ml) with a correlation coefficient greater than 0.99. The limit of detection was between 2.07×108 and 4.35×109 whereas the limit of quantification was between 6.90×108 and 1.45×1010VP/ml, respectively. The coefficient of variation of the intra- and inter-day precision of the method was less than 5% and 10%. HPLC data compared well with results obtained by electron microscopy, HA assay and with a virus counter, and was used to monitor virus concentrations in the supernatant obtained directly from the cell culture production vessels. The HPLC influenza virus analytical method can potentially be suitable as an in-process monitoring tool to accelerate the development of processes for the manufacturing of influenza vaccines.

Lot-to-lot consistency of live attenuated SA 14-14-2 Japanese encephalitis vaccine manufactured in a good manufacturing practice facility and non-inferiority with respect to an earlier product

We conducted a four-arm, double-blind, randomized controlled trial among 818 Bangladeshi infants between 10 and 12 months of age to establish equivalence among three lots of live attenuated SA 14-14-2 JE vaccine manufactured by the China National Biotec Group's Chengdu Institute of Biological Products (CDIBP) in a new Good Manufacturing Practice (GMP) facility and to evaluate non-inferiority of the product with a lot of the same vaccine manufactured in CDIBP's original facility.The study took place in two sites in Bangladesh, rural Matlab and Mirpur in urban Dhaka. We collected pre-vaccination (Day 0) and post-vaccination Day 28 (−4 to +14 days) blood samples to assess neutralizing anti-JE virus antibody titers in serum by plaque reduction neutralization tests (PRNT). Seroprotection following vaccination was defined as a PRNT titer ≥1:10 at Day 28 in participants non-immune at baseline. Follow-up for reactogenicity and safety was conducted through home visits at Day 7 and monitoring for serious adverse events through Day 28.Seroprotection rates ranged from 80.2% to 86.3% for all four lots of vaccine. Equivalence of the seroprotection rates between pairs of vaccine lots produced in the new GMP facility was satisfied at the pre-specified 10% margin of the 95% confidence interval (CI) for two of the three pairwise comparisons, but not for the third (−4.3% observed difference with 95% CI of −11.9 to 3.3%). Nevertheless, the aggregate seroprotection rate for all three vaccine lots manufactured in the GMP facility was calculated and found to be within the non-inferiority margin (within 10%) to the vaccine lot produced in the original facility. All four lots of vaccine were safe and well tolerated.These study results should facilitate the use of SA 14-14-2 JE vaccine as a routine component of immunization programs in Asian countries.