Design and development of a simple method for the detection and quantification of residual host cell DNA in recombinant rotavirus vaccine.

Rotavirus recombinant vaccine is usually produced in Vero cells. Residual host DNA may reside in the final product and is considered a source of contamination. WHO protocols indicate that biological products should be free of any type of impurity such as nucleic acids, endotoxins, and host cell intermediate materials. Therefore, all recombinant biological therapeutics should be assessed for residual host DNA. In the present study, a sensitive and specific real-time PCR method was developed to detect residual host cell DNA in the final product. The Beta-actin gene of Vero cells was selected to detect residual host cell DNA. One set of primers and a TaqMan probe were designed for the gene using AlleleID 6 software. Real-time PCR reactions were set up, and efficiency of 84% was obtained. The sensitivity and limit of detection of the assay were determined to be 0.176 Fg/µl and 0.044 Fg/µl, respectively. The intra-assay and inter-assay variations were 4.4% and 1.04%, respectively. Furthermore, the specificity and sensitivity of the assay were high enough, and the detection limit was lower than that of the FDA and WHO standards. This indicates that our assay is highly specific and sensitive to detect residual host DNA of Vero cells in the recombinant rotavirus vaccine.

Development of an imaged capillary isoelectric focusing method for characterizing the surface charge of mRNA lipid nanoparticle vaccines.

Lipid nanoparticles (LNPs) have been employed for drug delivery in small molecules, siRNA, mRNA, and pDNA for both therapeutics and vaccines. Characterization of LNPs is challenging because they are heterogeneous mixtures of large complex particles. Many tools for particle size characterization, such as dynamic and static light scattering, have been applied as well as morphology analysis using electron microscopy. CE has been applied for the characterization of many different large particles such as liposomes, polymer, and viruses. However, there have been limited efforts to characterize the surface charge of LNPs and CIEF has not been explored for this type of particle. Typically, LNPs for delivery of oligonucleotides contain at least four different lipids, with at least one being an ionizable cationic lipid. Here, we describe the development of an imaged capillary isoelectric focusing method used to measure the surface charge (i.e., pI) of an LNP-based mRNA vaccine. This method is capable of distinguishing the pI of LNPs manufactured with one or more different ionizable lipids for the purpose of confirming LNP identity in a manufacturing setting. Additionally, the method is quantitative and stability-indicating making it suitable for both process and formulation development.

Development of imaged capillary isoelectric focusing method and use of capillary zone electrophoresis in hepatitis B vaccine RECOMBIVAX HB®.

The hepatitis B virus vaccine consists of a major surface antigen called HBsAg, which is a lipid-bound protein that self-assembles into 22 nm spherical noninfectious virus-like particles (VLPs). The HBsAg VLP particles are expressed in yeast and have been well-characterized biochemically and biophysically employing various analytical techniques. In fact, a CZE method has been developed for monitoring process purification of the hepatitis B vaccine. Another CE-based method, imaged capillary IEF (icIEF) has been used extensively in the field of protein-based drug development as a tool for product identification,stability monitoring, and characterization. Here we describe the development of the icIEF method using the iCE280 instrument from ProteinSimple for measuring the pI and monitoring the profiles of HBsAg VLP particles. This method was applied to characterize the stability of the HBsAg VLP particles in three different formulation buffers. The results show that HBsAg VLP particles have a pI of 2.7 and it is one of most acidic particles that we have measured by icIEF. In addition to icIEF, we have also employed a CZE method to measure the electrophoretic mobility of HBsAg VLP particles and compared the results with icIEF and dynamic light scattering methods, showing consistent correlation among the three methods in terms of HBsAg VLP particles aggregation.

A (RP)UHPLC/UV analytical method to quantify dsRNA during the mRNA vaccine manufacturing process.

dsRNA is a product related impurity produced during the mRNA manufacturing process. The established immuno-based detection methods lack the flexibility and speed required to be applied throughout the manufacturing process. The RP-HPLC method developed outperforms these in terms of precision, broader detection range, LOD and LOQ, as well as in output variance. Using this method, dsRNA can be quantified in under 30 min for a single sample.

mRNA Vaccine Development for Emerging Animal and Zoonotic Diseases.

In the prevention and treatment of infectious diseases, mRNA vaccines hold great promise because of their low risk of insertional mutagenesis, high potency, accelerated development cycles, and potential for low-cost manufacture. In past years, several mRNA vaccines have entered clinical trials and have shown promise for offering solutions to combat emerging and re-emerging infectious diseases such as rabies, Zika, and influenza. Recently, the successful application of mRNA vaccines against COVID-19 has further validated the platform and opened the floodgates to mRNA vaccine's potential in infectious disease prevention, especially in the veterinary field. In this review, we describe our current understanding of the mRNA vaccines and the technologies used for mRNA vaccine development. We also provide an overview of mRNA vaccines developed for animal infectious diseases and discuss directions and challenges for the future applications of this promising vaccine platform in the veterinary field.

Streamlining Peptide Mapping LC-MS Approach for Studying Fusion Peptide-Conjugated Vaccine Immunogens.

A newly introduced HIV-1 vaccination utilizes a fusion peptide (FP)-based immunogen-carrier conjugate system, where the FP is coupled to a protein carrier via a bifunctional linker. Such heterogeneous materials present a challenge for the routine product quality assessment. Peptide mapping LC-MS analysis has become an indispensable tool for assessing the site-specific conjugation ratio, estimating site occupancy, monitoring conjugation profiles, and analyzing post-translational modifications (PTMs) and disulfide bonds as well as high-order protein structures. To streamline the peptide mapping approach to match the needs of a fast-paced conjugate vaccine product characterization, a selection of signature fragment ions generated by MSE fragmentation was successfully applied to assess the product quality at the different stages of a conjugates' manufacturing process with an emphasis on monitoring the amount of a reactive linker. This technique was employed in different conjugation studies of the protein carriers, linkers, and FP compositions as well as the cross-linked species formed during stress-degradation studies. Multiple derivatives of the intermediate and final conjugated products formed during a multistaged synthesis were monitored by means of the sensitive extracted-ion chromatogram (XIC) profiling and were included in the estimation of the site-specific conjugation loads. Differentiation of the conjugates with various FP compositions was demonstrated. The conjugation site occupancy was evaluated with respect to the solvent exposure of Lys residues. The findings of these LC-MS studies greatly aided in choosing the best conjugation strategy to ensure that the final recombinant tetanus toxoid heavy chain (rTTHc) product is chemically inert and represents a safe vaccine candidate for clinical evaluation.

Detection of virulence factors of South African Lactococcus garvieae isolated from rainbow trout, Oncorhynchus mykiss (Walbaum).

Lactococcus garvieae is a Gram-positive bacterium that causes mortalities in freshwater and marine fish worldwide and therefore results in severe economic losses in the aquaculture industry. Apart from the apparent integral role of the exopolysaccharide (EPS) capsule in pathogenesis, factors associated with virulence of this bacterium are poorly understood. However, recent studies have indicated that the ability of L. garvieae to cause disease does not depend on the presence of the EPS capsule. Lack of knowledge of virulence factors, pathogenesis and serology of L. garvieae is an impediment to the development of effective typing methods and control measures. This study, therefore, aimed to detect the presence of EPS capsules and other putative virulence factors in South African L. garvieae fish pathogenic isolates and a non-virulent isolate, and to identify possible candidates for subunit vaccine development. No indication of the presence of the EPS capsule was detected by negative staining or amplification of the EPS biosynthesis gene cluster in the virulent isolates or the avirulent strain, discrediting the notion that the EPS capsule is the sole determinant of virulence. However, a set of putative virulence factor genes was detected in all isolates, and candidates for subunit vaccine development (enolase, lactate dehydrogenase phosphoenolpyruvate-protein phosphotransferase) were identified by identification of extracellular proteins of virulent strains.

Detection of classical swine fever vaccine virus in blood and tissue samples of pigs vaccinated either with a conventional C-strain vaccine or a modified live marker vaccine.

Attenuated live classical swine fever (CSF) viruses are the most efficacious vaccines against the disease. However, little is known about the distribution and detection of CSF vaccine viruses in the host. We therefore compared the new recombinant attenuated marker vaccine virus CP7_E2alf with the conventional C-strain vaccine concerning virus isolation, antigen-, and genome-detection in different samples within the first 42 days post-vaccination (p.v.). Leukocytes and several organs such as tonsils, lymph nodes, spleen, thymus, parotis and kidney were also tested using highly sensitive real-time reverse transcription-polymerase chain reaction (RT-PCR) techniques. It was demonstrated that vaccine virus could be detected by live animal sampling only in a few leukocytes samples at very low titres and genome copy numbers within the first 14 days after immunisation. Vaccine virus could also be isolated from individual tonsil samples within the first 6 days after vaccine application. In contrast, vaccine virus genomes were consistently detected in the tonsils up to day 42 by real-time RT-PCR. Distribution, amount of virus and viral genome levels were similar for both tested vaccines. In conclusion, blood samples could be the sample material of choice for detecting CSF wild type virus infection even in vaccinated animals after more than 14 days p.v., while tonsil sampling provided appropriate material for long-term detection of both tested CSF vaccine viruses using real-time RT-PCR methods.

[Clinical trials of oral recombinant bivaccine against variola and hepatitis B during double vaccination].

Clinical trials of oral live recombinant embryonic variola and hepatitis B bivaccine as tablets (Revax-BT) were performed. When volunteers were prevaccinated with oral variola vaccine first in a small dose and, 7, 14, 30, 90, and 180 days later, in a larger dose, a slight reactoginicity was sometimes observed after the first vaccination (with a small dose) whereas revaccination with a larger dose did not give rise to any clinical manifestations. A month after vaccination, a protective level of virus-neutralizing antibodies to vaccinia virus (VV) was observed in 90-100% of the volunteers twice immunized with the bivaccine (in a small dose and in a larger one at an administration intervals of 1-2 weeks under remote revaccination while 6-9 months following vaccination, this level was recorded in 80% of the volunteers. A month following vaccination, 50-55% seroconversion to VV was observed in the volunteers twice immunized with the bivaccine (at an interval of 1 or 3-6 months). Cellular immunity to VV was low (0-20%). Double immunization of volunteers with the oral bivaccine under remote vaccination failed to produce the significant levels of humoral and cellular immune responses to hepatitis B markers. Recombinant VV was not recorded in any blood, saliva, and urine samples taken in the volunteers twice immunized with the bivaccine.

Quantitation of putative glycoprotein X in bioengineered pseudorabies vaccine virus culture medium by ELISA.

An enzyme-linked immunosorbent assay has been developed for the detection and quantitation of putative pseudorabies glycoprotein X (gX) in bulk bioengineered PRV delta gX delta tk-1 pseudorabies vaccine virus culture medium supernatants. The assay has a dynamic range of 0.2-25 ng, with a best linear region of 0.4-12.5 ng (correlation coefficient = 0.99) which permits 1 ppm discrimination for gX.

Development of a quantitative assay for residual host cell proteins in a recombinant subunit vaccine against human respiratory syncytial virus.

We have developed and validated a process-specific immunoligand assay based on the Threshold system for the quantification of residual host cell proteins (HCPs) in a recombinant subunit vaccine candidate against the human respiratory syncytial virus (hRSV). The industrial process of this vaccine produced in Escherichia coli, involved five chromatography steps for the production of clinical-grade batches. The clearance of non-product-related protein throughout the purification process was documented by the evaluation of the HCP content in the chromatographic fractions at each step of the downstream processing. The assay had a detection limit of 0.5 ng/ml of HCP equivalent to 10 parts per million (ppm). The quantification limit was 1.3 ng/ml of HCP, giving a sensitivity range of the assay of 10 to 30 ppm. To our knowledge, this is the first sensitive HCP assay reported for a vaccine.

Studies in owl monkeys leading to the development of a synthetic vaccine against the asexual blood stages of Plasmodium falciparum.

During the development of a synthetic vaccine for human use against the asexual blood stages of Plasmodium falciparum, monkey trials were performed to assess safety, immunogenicity, and protectivity. We determined the minimal infective dose of the P. falciparum FVO strain, the kinetics of the immune response induced by vaccination with the synthetic peptide mixture (S7 + S12 + S17) or the synthetic hybrid polymeric protein SPf66, and the induction of protective immunity against the experimental challenge with 2 P. falciparum strains. A clear boosting effect was observed, determined by the increased antibody titers against synthetic peptides S7, S12, S17, and SPf66, and by improvement in the protective immune response against the challenge. These studies suggest that either the peptide mixture or the synthetic hybrid polymeric protein are excellent choices for the development of a vaccine against P. falciparum.

In vitro assessment of viral antigen content in inactivated aluminum hydroxide adjuvanted vaccines.

Antigen capture enzyme immunoassays (ELISA) were developed to assess the antigenic content of inactivated aluminum hydroxide (AH) adjuvanted porcine parvovirus, pseudorabies, and infectious bovine rhinotracheitis vaccines. Reference preparations of these viruses were constructed as a basis for comparison. Because AH-associated ELISA interference was largely circumvented, the need for isotopic or complex antigen-adjuvant desorption methods was eliminated. A 4-parameter logistic model related optical density to vaccine dilution. High correlation coefficients (r) were routinely achieved with commercial monovalent and polyvalent vaccines, and reference preparations. The procedure quantified antigen in both aqueous and AH-associated phases. The method may be generally applicable as a partial substitute for in vivo vaccine potency testing by allowing in vitro estimation of inactivated viral antigenic content in AH adjuvanted vaccines.

Reversed-phase high-performance liquid chromatographic study of thimerosal stability in Cuban recombinant hepatitis B vaccine.

Non-degraded thimerosal was determined in the presence of its decomposition products by directly assaying recombinant hepatitis B vaccine using a reversed-phase liquid chromatographic method. Methanol-water-orthophosphoric acid (65:35:0.9, v/v/v) was used as the eluent. Salicylic acid was employed as an internal standard. The calibration graph was linear (r = 0.99995) up to 2.5 micrograms of thimerosal. Interference from aluminium hydroxide was eliminated by centrifugation. Good stability of thimerosal in the hepatitis B vaccine was demonstrated. The results obtained were in agreement with the recently proposed mechanism of degradation.

Host cell contaminant protein assay development for recombinant biopharmaceuticals.

The efficiency and consistency of a biopharmaceutical purification process determines drug quality, including which specific types and concentrations of residual host cell or process contaminants may remain. Commercial reagents and generic analytical methods are available for quantitating most of these contaminants. However, no generic assay is available for quantitation of the specific contaminant host cell proteins (HCPs) which are unique to a novel purification process. Because of this, proprietary reagents and assays must be developed for the quantitation of process-specific HCPs in each biopharmaceutical drug. The need to develop proprietary reagents which are both sensitive to, and specific for, potentially complex mixtures of unique contaminant proteins has defined what is acceptable methodology for development of quantitative HCP assays. Within the biopharmaceutical industry this need is most often satisfied by the development of multi-analyte HCP immunoassays based upon the null cell mock purification model. Confidence in the quantitative nature of a given HCP assay, and the validity of analytical measurement obtained by the assay, is dependent upon empirical demonstration of the unique stoichiometry of the HCP assay reagents. In conjunction with other analytical and validation methods, an HCP immunoassay may be thought of as a necessary quantitative tool for the optimization and validation of biopharmaceutical purification process efficiency and consistency, rather than as an end in itself.

Insect cell-derived VP2 of infectious bursal disease virus confers protection against the disease in chickens.

Infectious bursal disease virus (IBDV) has become a major problem in recent years. Conventional vaccines make use of attenuated or inactivated viral strains, but these are gradually losing their effectiveness. We investigated the possibility of using purified VP2, a subunit of IBDV structural protein expressed in insect cells, as a vaccine. The VP2 gene was cloned into pAcYM1. The cloned gene was expressed in a baculovirus system, giving rise to a high quantity of recombinant VP2 (rVP2) protein. The length of the VP2 is 453 amino acids, and it contains two additional amino acids of the baculovirus at the carboxyl terminus. The molecular mass of the protein is about 48 kD. The rVP2 protein reacted with antibodies raised against viral VP2 and had a similar molecular weight. This protein was tested in a controlled vaccination experiment and compared with an inactivated commercial vaccine. High levels of antibodies were raised by the vaccinated birds. The vaccinated birds were challenged with a pathogenic viral strain. rVP2-vaccinated chickens exhibited high resistance to the virus. No mortality or weight changes in the bursa of Fabricius were observed in the vaccinated birds, whereas in the negative control birds, vaccinated with phosphate buffer, up to 50% mortality was found. Higher levels of antibodies were found by enzyme-linked immunosorbent assay in birds vaccinated with rVP2 compared with those vaccinated with the commercial vaccine. This study suggests the potential use of the isolated rVP2 as a subunit vaccine.

Onset of protective immunity in chicks after vaccination with a recombinant herpesvirus of turkeys vaccine expressing Newcastle disease virus fusion and hemagglutinin-neuraminidase antigens.

The onset of protective immunity from lethal Newcastle disease virus (NDV) challenge of chicks was determined after vaccination with a recombinant herpes virus of turkeys (HVT) expressing the fusion and hemagglutinin-neuraminidase proteins of NDV. One-day-old specific-pathogen-free chicks devoid of maternal antibodies to NDV were vaccinated with 130 to 3300 plaque forming units of HVT (depending on the trial) and then challenged at 4, 7, 10, and 14 days postvaccination (DPV) with a neurotropic velogenic strain of NDV (GB Texas). The recombinant vaccine afforded 0%, 35-75%, 85%, and 94-100% protection when the vaccinated birds were challenged at 4, 7, 10, and 14 DPV, respectively. In all trials, challenge caused 100% mortality in unvaccinated control chicks. Newcastle disease virus was reisolated from the lung, liver, spleen, and brain of birds dying in all trials regardless of vaccine dosage or time of challenge, except when challenge occurred at 14 DPV.

Successful pseudorabies vaccination in maternally immune piglets using recombinant vaccinia virus vaccines.

Three gilts were vaccinated with a NYVAC vaccinia recombinant expressing glycoprotein gD of pseudorabies virus (PRV) (NYVAC/gD). After farrowing, the piglets were allowed to nurse normally to obtain colostral immunity and then were divided into four groups, receiving NYVAC/gD, a NYVAC recombinant expressing glycoprotein gB of PRV (NYVAC/gB), an inactivated PRV vaccine (iPRV), or no vaccine. The piglets were vaccinated twice, three weeks apart beginning at approximately two weeks of age and later challenged with virulent PRV oronasally. Piglets that received NYVAC/gB or iPRV were the best protected based on lack of mortality, lower temperature responses, decreased weight loss and decreased viral shedding after challenge. These results indicate effective strategies for stimulating active immune response while still under the protection of maternal immunity.

Protection of cattle and swine against foot-and-mouth disease, using biosynthetic peptide vaccines.

A single dose of foot-and-mouth disease (FMD) virus protein 1 (VP1) peptide, expressed in Escherichia coli as a fusion protein with 190 amino acids (AA) of the LE' protein of the tryptophan operon of E coli, elicited an immune response in steers sufficient to withstand the challenge of exposure to animals with acute FMD. The 58-micrograms dose of viral peptide, composed of a segment of the VP1 from the A12 strain (A12) of FMD virus (FMDV; A12-32dimer) in a tandem repeat configuration of AA137 through 168 and emulsified with oil adjuvant, elicited a serologic response in cattle equivalent to that obtained using conventional whole virus vaccines. Two groups of swine were vaccinated, 1 with the A12-32dimer as used in cattle and 1 with AA131 through 157 from VP1 of the A24 strain (A24) of FMDV (A24-peptide), expressed in the same system as A12-32dimer, but as a single copy per molecule. In swine, the 58-micrograms dose of the A12-32dimer repeated at 28 days was an effective immunogen; all swine were protected against A12 and, in addition, the vaccine protected 50% of the swine against A24. The 29-micrograms dose of A24-peptide, administered according to the same schedule, elicited protection against A24 in 50% of the vaccinates and, in addition, protected 25% of those vaccinates against A12. The serologic response elicited by A12-32dimer against A24 virus was considerably greater than the response elicited by A24-peptide against A12 virus. The evidence of multiple immunogenic epitopes between AA131 and AA168 was evaluated.

Identification of an immunodominant region on the isolated bovine leukaemia virus (BLV) major envelope protein gp51 by monoclonal antibodies presumably not exposed during natural BLV infection.

A panel of newly isolated monoclonal antibodies (MoAbs) is described which are specific for bovine leukaemia virus (BLV) envelope protein gp51. Epitope mapping using competition antibody binding assays and binding studies with gp51-related fusion proteins and synthetic peptides show that they are directed against seven independent antigenic determinants. Four of them are unrelated to the epitopes described earlier (Bruck et al., 1982a). We define three binding regions for the MoAbs on the gp51 molecule. The region between amino acids 170 and 217 is highly immunogenic when the isolated protein is used for immunization, and seems to be inaccessible for immune recognition when gp51 is associated with the virus envelope as it occurs during natural BLV infection.