Development of a droplet digital PCR for pertussis toxin locus copy number determination in a genetically-modified Bordetella pertussis strain.

To improve pertussis toxin (PT) yield in B. pertussis strains for vaccine production a genetically-engineered strain (gdPT 191-134 strain) with a second copy of the genetically detoxified PT (gdPT) locus was developed. The consistency of the production and genetic stability of the strain when used for vaccine production must be established. We developed two simplex ddPCR assays with PCR systems for ptxA, the target gene present in two copies, and pgm, the reference gene present as a single copy. The ddPCR assay had sufficient precision to discriminate the copy number of the PT locus accurately in two B. pertussis strains: one copy in the parent, non-genetically-engineered strain and two copies in the gdPT 191-134 strain. Using the ddPCR assays, we were able to show that the ratio of the ptxA to pgm genes decreased during serial culture passages, due to the loss of PT locus, which in turn, resulted in lower levels of PT production over time. We were then able to assess culture conditions that improved the stability of the double locus, as shown by non-significant reduction in gdPT toxin yield.

Multiplex reverse transcriptase droplet digital PCR for the simultaneous quantification of four dengue serotypes: Proof of concept study.

During vaccine production, RNA from chimeric yellow fever-dengue (CYD) vaccine viruses (CYD1, CYD2, CYD3 and CYD4) is currently quantified using separate serotype-specific RT-qPCR assays. Here we describe the results from a proof-of-concept study on the development of a multiplex reverse transcriptase droplet digital PCR (RT-ddPCR) assay for simultaneous quantification of RNA for all four viruses. Serotype-specific simplex RT-ddPCRs were developed using the serotype-specific PCR systems (forward and reverse primers and FAM (fluorescent chromophores 6-carboxyfluorescein) and YY (Yakima Yellow)-labelled probes), used in the routine RT-qPCR. The PCR systems were specific and gave similar quantification results to those from the RT-qPCR assay. Linear regression analyses were used to select relative probe concentrations to obtain distinct clusters for each target RNA in a 2-D cluster plot in a multiplex RT-ddPCR assay. We showed the clusters were positioned as predicted in the model for each CYD RNA and were well separated. The multiplex RT-ddPCR gave similar quantification results to those obtained by the serotype-specific RT-qPCR assays for triplicate samples containing 7, 8 or 9 Log10 Geq/mL. In conclusion, these results demonstrate that it is possible to quantify RNA from four CYD serotypes with a multiplex RT-ddPCR assay in a single assay.

Host cell protein testing strategy for hepatitis B antigen in Hexavalent vaccine - Towards a general testing strategy for recombinant vaccines.

BACKGROUND: Recombinant proteins expressed in host cell systems may contain host cell proteins (HCP) as impurities. While there is no clear evidence of clinical adverse events attributable to HCP, HCP levels and profiles must be documented to meet regulatory requirements and to understand the consistency of the biological product and manufacturing process. We present a general strategy for HCP characterization applied to a recombinant protein antigen, Hepatitis B surface antigen (HBsAg) used in a multivalent vaccine. METHODS: Polyclonal antisera raised against HCPs in process fractions from a mock preparation of the HBsAg yeast expression host, Hansenula polymorpha, were used to develop a quantitative sandwich ELISA to measure HCP content in batches of purified recombinant HBsAg. Batches were also subjected to SDS-PAGE and LC-MS/MS to identify detectable proteins. Batch consistency was further assessed by SDS-PAGE/densitometry purity analysis and by the ratio of specific HBsAg content (by ELISA) to total protein. RESULTS: Using the quantitative HCP ELISA, the HCP content showed no discernable trend in multiple HBsAg batches manufactured over a 5-year period. All batches were ≥95% pure by SDS-PAGE/densitometry, with consistent HBsAg/total protein ratios. In addition to the expected HBsAg antigen protein, LC-MS/MS analysis of three HBsAg batches identified several yeast proteins, none of which are known to cause adverse reactions in humans. CONCLUSIONS: Analysis of multiple HBsAg batches showed consistent HCP content and identification profiles, as well as product purity and specific antigen content, demonstrating consistent manufacturing process. Recombinant vaccines, unlike therapeutic products, are administered infrequently with only small amounts of protein injected at a time. With limited potential for adverse reactions to small quantities of HCPs in purified recombinant vaccine antigens, and considering the relevant regulatory guidelines, we conclude that once consistent manufacturing process has been demonstrated, routine HCP testing in recombinant vaccine antigens is no longer required.

Innovative in cellulo method as an alternative to in vivo neurovirulence test for the characterization and quality control of human live Yellow Fever virus vaccines: A pilot study.

Live attenuated vaccines have proved to be mostly valuable in the prevention of infectious diseases in humans, especially in developing countries. The safety and potency of vaccine, and the consistency of vaccine batch-to-batch manufacturing, must be proven before being administrated to humans. For now, the tests used to control vaccine safety largely involve animal testing. For live viral vaccines, regulations require suppliers to demonstrate the absence of neurovirulence in animals, principally in non-human primates and mice. In a search to reduce the use of animals and embracing the 3Rs principles (Replacement, Reduction, Refinement in the use of laboratory animals), we developed a new Blood-Brain Barrier Minibrain (BBB-Minibrain) in cellulo device to evaluate the neuroinvasiveness/neurovirulence of live Yellow Fever virus (YFV) vaccines. A pilot study was performed using the features of two distinct YFV strains, with the ultimate goal of proposing a companion test to characterize YFV neurovirulence. Here, we demonstrate that the BBB-Minibrain model is a promising alternative to consider for future replacement of YFV vaccine in vivo neurovirulence testing (see graphical abstract).

Comparison of reverse-transcriptase qPCR and droplet digital PCR for the quantification of dengue virus nucleic acid.

Polymerase chain reaction (PCR) is an important molecular biology technique for in vitro amplification of nucleic acids. Reverse transcriptase quantitative PCR (RT-qPCR) and more recently reverse transcriptase digital droplet PCR (RT-ddPCR) have been developed for the quantification of nucleic acids. We developed an RT-ddPCR assay for the quantification of attenuated dengue virus serotype 2 nucleic acid and compared it with a routine RT-qPCR assay. While the routine RT-qPCR assay targets the NS5 gene, the E gene was selected for the optimization of the RT-ddPCR assay conditions. The specificity of the assay was demonstrated using the attenuated dengue virus serotype 2 alone and in the presence of the other three dengue serotypes. The results from both assays for 25 samples of the attenuated dengue virus serotype 2 were found to be comparable, with an R2 from the linear regression analysis of >0.98. A major advantage of the RT-ddPCR assay is that it allows quantification of nucleic acid, without the need of a standard curve. RT-ddPCR can be implemented for the absolute quantification of dengue vaccine virus nucleic acid during the vaccine manufacturing process.

A latex agglutination assay to quantify the amount of hemagglutinin protein in adjuvanted low-dose influenza monovalent vaccines.

To formulate inactivated influenza vaccines, the concentration of hemagglutinin (HA) must be accurately determined. The standard test currently used to measure HA in influenza vaccines is the Single Radial Immunodiffusion (SRID) assay. We developed a very rapid, simple and sensitive alternative quantitative HA assay, namely the Latex Agglutination Assay (LAA). The LAA uses the Spherotest® technology, which is based on the agglutination of HA-specific immunoglobulin-coated latex beads. The amount of HA in a sample is calculated from the level of bead agglutination by a simple absorbance measurement at 405nm against a standard curve generated using a monovalent vaccine standard. In less than 2hours, tens of samples could be quantified using the LAA as opposed to 2days for the SRID assay. Ten steps are required to complete an SRID assay as compared to 6 steps for the LAA, from sample preparation through spectrophotometric analysis. Furthermore, the limit of detection of the LAA was found to be approximately 15ng HA/mL, similar to an ELISA, with the quantification of less than 1.8µg HA/mL. The quantification limit of the SRID is usually considered to be approximately 5µg HA/mL. The development of the assay and a comparison of the titers obtained by SRID and LAA for several monovalent vaccines corresponding to various strains were performed. For A/H5N1 and A/H1N1 monovalent vaccines, the LAA was found to be linear and accurate as compared to the SRID. The precision of the LAA was close to that of the standard test, and good reproducibility from one laboratory to another was observed. Moreover, the LAA enabled HA quantification in AlOOH-adjuvanted and in emulsion-adjuvanted low-dose vaccines as well as unadjuvanted vaccines. In conclusion, LAA may be useful to rapidly and accurately measure influenza HA protein in monovalent vaccines, especially in those containing less than 5µg/mL of HA in the presence of an adjuvant.

Nine µg intradermal influenza vaccine and 15 µg intramuscular influenza vaccine induce similar cellular and humoral immune responses in adults.

Intanza® 9 µg (Sanofi Pasteur), a trivalent split-virion vaccine administered by intradermal (ID) injection, was approved in Europe in 2009 for the prevention of seasonal influenza in adults 18 to 59 years. Here, we examined the immune responses induced in adults by the ID 9 µg vaccine and the standard trivalent intramuscular (IM) vaccine (Vaxigrip® 15 µg, Sanofi Pasteur). This trial was a randomized, controlled, single-center, open-label study in healthy adults 18 to 40 years of age during the 2007/8 influenza season. Subjects received a single vaccination with the ID 9 µg (n=38) or IM 15 µg (n=42) vaccine. Serum, saliva, and peripheral blood mononuclear cells were collected up to 180 days post-vaccination. Geometric mean hemagglutination inhibition titers, seroprotection rates, seroconversion rates, and pre-vaccination-to-post-vaccination ratios of geometric mean hemagglutination inhibition titers did not differ between the two vaccines. Compared with pre-vaccination, the vaccines induced similar increases in vaccine-specific circulating B cells at day 7 but did not induce significant increases in vaccine-specific memory B cells at day 180. Cell-mediated immunity to all three vaccine strains, measured in peripheral blood mononuclear cells, was high at baseline and not increased by either vaccine. Neither vaccine induced a mucosal immune response. These results show that the humoral and cellular immune responses to the ID 9 µg vaccine are similar to those to the standard IM 15 µg vaccine.

Humoral and cellular immune responses to split-virion H5N1 influenza vaccine in young and elderly adults.

We evaluated the humoral and cellular immunogenicity of adjuvanted and non-adjuvanted H5N1 influenza vaccine in two groups of 300 adults: aged 18-60 and >60 years in a randomized, open-label, uncontrolled phase 2 trial. Participants received two injections (D0, D21) of 7.5 microg hemagglutinin without adjuvant or 30 microg with aluminum hydroxide adjuvant. Antibody responses and cytokine secretion were assessed before and after vaccination. Excluding the 6/300 non-elderly and 47/300 elderly participants with pre-existing antibodies, geometric mean titers (dil(-1)) on D42 were higher with 30 microg+Ad and were comparable between age groups. Participants with pre-existing antibodies responded strongly to the first vaccination (GMTs in the range 147-228 on D21). Vaccination increased both Th1 and Th2 T-cell responses. The predominantly Th1 profile observed before vaccination was unaffected by vaccination. H5N1 influenza vaccine is no less immunogenic in elderly adults than in younger adults and, due to a higher proportion non-naïve elderly, immunogenicity was higher in this latter group.

Safety, humoral and cell mediated immune responses to two formulations of an inactivated, split-virion influenza A/H5N1 vaccine in children.

BACKGROUND: Highly pathogenic influenza A/H5N1 has caused outbreaks in wild birds and poultry in Asia, Africa and Europe. It has also infected people, especially children, causing severe illness and death. Although the virus shows limited ability to transmit between humans, A/H5N1 represents a potential source of the next influenza pandemic. This study assesses the safety and immunogenicity of aluminium hydroxide adjuvanted (Al) and non adjuvanted influenza A/Vietnam/1194/2004 NIBRG-14 (H5N1) vaccine in children. METHODS AND FINDINGS: In a Phase II, open, randomised, multicentre trial 180 children aged 6 months to 17 years received two injections, 21 days apart, of vaccine containing either: 30 microg haemagglutinin (HA) with adjuvant (30 microg+Al) or 7.5 microg HA without adjuvant. An additional 60 children aged 6-35 months received two "half dose" injections (ie 15 microg+Al or 3.8 microg). Safety was followed for 21 days after vaccination. Antibody responses were assessed 21 days after each injection and cellular immune responses were explored. Vaccination appeared well tolerated in all age groups. The 30 microg+Al formulation was more immunogenic than 7.5 microg in all age groups: in these two groups 79% and 46% had haemagglutinination inhibition antibody titres > or =32 (1/dil). Among 6-35 month-olds, the full doses were more immunogenic than their half dose equivalents. Vaccination induced a predominantly Th2 response against H5 HA. CONCLUSIONS: This influenza A(H5N1) vaccine was well tolerated and immunogenic in children and infants, with Al adjuvant providing a clear immunogenic advantage. These results demonstrate that an H5N1 Al-adjuvanted vaccine, previously shown to be immunogenic and safe in adults, can also be used in children, the group most at risk for pandemic influenza.

Cell-mediated immunity induced by chimeric tetravalent dengue vaccine in naive or flavivirus-primed subjects.

Three independent, phase 1 clinical trials were conducted in Australia and in USA to assess the safety and immunogenicity of sanofi pasteur dengue vaccine candidates. In this context, Dengue 1-4 and Yellow Fever 17D-204 (YF 17D)-specific CD4 and CD8 cellular responses induced by tetravalent chimeric dengue vaccines (CYD) were analyzed in flavivirus-naive or flavivirus-immune patients. Tetravalent CYD vaccine did not trigger detectable changes in serum pro-inflammatory cytokines, whatever the vaccinees immune status, while inducing significant YF 17D NS3-specific CD8 responses and dengue serotype-specific T helper responses. These responses were dominated by serotype 4 in naive individuals, but a booster vaccination (dose #2) performed 4 months following dose #1 broadened serotype-specific responses. A similar, broader response was seen after primary tetravalent immunization in subjects with pre-existing dengue 1 or 2 immunity caused by prior monovalent live-attenuated dengue vaccination. In all three trials, the profile of induced response was similar, whatever the subjects' immune status, i.e. an absence of Th2 response, and an IFN-gamma/TNF-alpha ratio dominated by IFN-gamma, for both CD4 and CD8 responses. Our results also showed an absence of cross-reactivity between YF 17D or Dengue NS3-specific CD8 responses, and allowed the identification of 3 new CD8 epitopes in the YF 17D NS3 antigen. These data are consistent with the previously demonstrated excellent safety of these dengue vaccines in flavivirus-naive and primed individuals.

Delivery of the HIV-1 Tat protein to dendritic cells by the CyaA vector induces specific Th1 responses and high affinity neutralizing antibodies in non human primates.

The human immunodeficiency virus type 1 (HIV-1) Tat is a key protein playing a major role in the infectivity of the virus. Thus, HIV-Tat based vaccines have been proposed as an attractive option to treat AIDS. Recently, we have shown that the recombinant detoxified adenylate cyclase (CyaA) from Bordetella pertussis carrying HIV-Tat (CyaA-E5-Tat), targets dendritic cells (DCs) and induces specific Th1 polarized and neutralizing antibody responses in mice. To further explore the potentialities of this prototype vaccine for human use, we analyzed the CyaA-E5-Tat induced antibody responses in non-human primates and established the biological characteristics of these antibodies. African Green Monkeys (AGM) were immunized with CyaA-E5-Tat in the presence or in the absence of alum adjuvant. First, we showed that the anti-CyaA antibodies induced by such immunization does not interfere with the binding of CyaA-E5-Tat to its receptor at the DC surface, the alphaMbeta2 integrin. Monkeys immunized with CyaA-E5-Tat, with or without alum, produced anti-Tat antibodies that mainly recognized the N-terminal domain of the Tat protein. Importantly, all sera obtained after three immunizations displayed the capacity to bind to Tat and neutralize its transactivating function in vitro. Finally, in the absence of alum, CyaA-E5-Tat, induced Th1 Tat specific T cell responses. These findings reveal that CyaA-E5-Tat is efficiently delivered in non-human primates and had a significant impact on the generation of neutralizing anti-Tat antibodies. These observations are, thus, encouraging for the use of the CyaA vector in human and also suggest that CyaA-E5-Tat might be a useful tool to decipher the biological characteristic of such antibodies.

Standardization of cytokine flow cytometry assays.

BACKGROUND: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). RESULTS: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells. CONCLUSION: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.