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.

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.

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.

Avaliação da imunogenicidade de diferentes formas alélicas da proteí­na recombinante PvAMA-1expressa em Pichia pastoris: impacto da diversidade antigênica / Evaluation of the immunogenicity of different allelic forms of PvAMA-1 protein expressed in Pichia pastoris: impact of antigenic diversity

A malária é um problema de saúde pública no Brasil e no mundo. Em 2016, o número de casos estimado pela Organização Mundial de Saúde foi de 216 milhões. Plasmodium falciparum é a espécie mais prevalente e responsável pelo maior número de mortes no mundo, sobretudo no continente africano. Por outro lado, o Plasmodium vivax é conhecido por sua ampla distribuição geográfica, sendo a espécie que predomina nas Américas, incluindo o Brasil. Nos últimos 20 anos, nosso grupo tem gerado e caracterizado diversas proteínas recombinantes baseadas em antígenos imunodominantes de P. vivax que podem servir como base para o desenvolvimento de uma vacina contra malária. Entre os antígenos de merozoítas, uma das principais proteínas em estudo pelo nosso grupo é o Antígeno 1 de Membrana Apical de P. vivax (PvAMA-1), caracterizado previamente como altamente imunogênico em infecções naturais e em camundongos imunizados, na presença de diferentes adjuvantes. O objetivo do presente estudo foi investigar o efeito da diversidade antigênica dessa proteína no reconhecimento por anticorpos específicos e na indução de imunidade contra o parasita. Para isso, foram geradas seis novas proteínas representando diferentes alelos descritos na natureza: PvAMA-1-Belem, PvAMA-1-Sal-I, PvAMA-1-Chesson-I, PvAMA-1-SK0814-apical, PvAMA-1-Indonesia-XIX e PvAMA-1-PNG_62_MU. As proteínas recombinantes foram expressas em leveduras Pichia pastoris e purificadas em duas etapas cromatográficas. Em seguida, as imunizações em camundongos C57BL/6 foram realizadas com as proteínas administradas de forma isolada, ou em combinação, na presença do adjuvante agonista de TLR3 (Poly I:C). Por ELISA, observamos que todas as formulações foram capazes de induzir anticorpos IgG contra as proteínas homólogas e heterólogas, o que sugere que a diversidade antigênica entre as formas alélicas não compromete o reconhecimento. Os dados gerados no presente trabalho sugerem que uma formulação contendo mistura de diferentes alelos representando a proteína AMA-1 pode ser explorada para o desenvolvimento de uma vacina de ampla cobertura contra o P. vivax

Malaria is a public health problem in Brazil and throughout the world. In 2016, the World Health Organization estimated there were 216 million cases of malaria. Plasmodium falciparum is the most prevalent species and is responsible for the largest number of deaths, especially in the African continent. However, Plasmodium vivax is known for its wide geographic distribution, being the species that prevails in the Americas, including Brazil. In the last 20 years, our group has generated and characterized several recombinant proteins based on immunodominant antigens of P. vivax that can serve as a basis for the development of a malaria vaccine. Among the merozoite antigens, one of the main proteins studied by our group is P. vivax apical membrane antigen-1 (PvAMA-1), previously characterized as highly immunogenic in natural infections and immunized mice, in the presence of different adjuvants. The objective of this study was to investigate the effect of antigenic diversity of this protein in the recognition of specific antibodies and the induction of immunity against the parasite. For this, six new proteins were generated representing different alleles described in nature: PvAMA-1-Belem, PvAMA-1-Sal-i, PvAMA-1-Chesson-i, PvAMA-1-SK0814-apical, PvAMA-1-Indonesia-XIX, and PvAMA-1-PNG_62_MU. Recombinant proteins were expressed in Pichia pastoris yeast and purified by two chromatographic stages. Then, C57BL/6 mice were immunized with these proteins administered in isolation or in combination, in the presence of the TLR3 agonist adjuvant, Poly I:C. Using an enzyme-linked immunosorbent assay, we observed that all formulations induced IgG antibodies against homologous and heterologous proteins. This indicates that antigenic diversity between allele forms does not compromise recognition. This finding suggests that a formulation containing a mixture of different alleles representing the PvAMA-1 protein can be exploited for developing of a wide coverage vaccine against P. vivax

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.

Characterization of a recombinant influenza vaccine candidate using complementary LC-MS methods.

Influenza vaccination is recognized as the most effective method for reducing morbidity and mortality due to seasonal influenza. To improve vaccine supply and to increase flexibility in vaccine manufacturing, cell culture-based vaccine production has emerged to overcome limitations of egg-based production. The switch of production system and the need for annual re-evaluation of vaccines for the effectiveness due to frequent viral antigenic changes call for methods for complete characterization of the hemagglutinin (HA) antigens and the final vaccine products. This study describes advanced liquid chromatography-mass spectrometry (LC-MS) methods for simultaneous identification of HA proteins and process-related impurities in a trivalent influenza candidate vaccine, comprised of purified recombinant HA (rHA) antigens produced in an insect cell-baculovirus expression vector system (BEVS). N-linked glycosylation sites and glycoforms of the three rHA proteins (corresponding to influenza A subtypes H1N1 and H3N2 and B virus, respectively) were profiled by peptide mapping using reversed-phase (RP) LC-MS(E) (data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode). The detected site-specific glycoforms were further confirmed and quantified by hydrophilic interaction LC (HILIC)-multiple reaction monitoring (MRM) assays. LC-MS(E) was used to characterize the vaccine candidate, providing both protein identities and site-specific information of glycosylation and degradations on each rHA protein. HILIC-MRM methodology was used for rapid confirming and quantifying site-specific glycoforms and potential degradations on each rHA protein. These methods can contribute to the monitoring of vaccine quality especially as it pertains to product comparability studies to evaluate the impact of production process changes.

Establishment and validation of an ELISA for the quantitation of HBsAg in recombinant hepatitis B vaccines.

Commercial enzyme-linked immunosorbent assay (ELISA) kits for the determination of the in vitro potency of recombinant hepatitis B vaccines, which detect hepatitis B surface antigen (HBsAg), have been used frequently as an alternative for traditional in vivo potency tests. With the constant need for validation procedures, an ELISA that could be employed to determine the in vitro potency of five recombinant hepatitis B vaccines simultaneously was established using two monoclonal antibodies. The use of two monoclonal antibodies produced "in house" specific for the small envelope protein S of the hepatitis B virus (HBV) resulted in the production of a highly specific, sensitive and stable ELISA. The standard ELISA parameters used in this study, considering the HBsAg content of each recombinant hepatitis B vaccine evaluated, resulted in a standard curve that could be applied for potency evaluations of different, commercial hepatitis B vaccine lots.

Evaluation of chemical degradation of a trivalent recombinant protein vaccine against botulinum neurotoxin by LysC peptide mapping and MALDI-TOF mass spectrometry.

Vaccines utilizing recombinant protein antigens typically require an adjuvant to enhance immune response in the recipients. However, the consequences of antigen binding to adjuvant on both the short- and long-term stability of the protein remain poorly defined. In our companion paper (Vessely et al., in press, J Pharm Sci), we characterized the effects of binding to adjuvant on the conformation and thermodynamic stability of three antigen variants for botulinum vaccines: rBoNTA(H(c)), rBoNTB(H(c)), and rBoNTE(H(c)). In the current study, we evaluated the effect of binding to adjuvant (Alhydrogel, aluminum hydroxide) on chemical stability of these antigens during long-term storage in aqueous suspension. We developed methods that employ LysC peptide mapping in conjunction with MALDI-TOF mass spectrometry. Peptide maps were developed for the proteins for a vaccine formulation of rBoNTE(H(c)) as well as a trivalent rBoNT(H(c)) vaccine formulation. This method provided high sequence coverage for the proteins in part due to the implementation of a postdigestion elution fractionation method during sample preparation, and was also successfully utilized to evaluate the chemical integrity of adjuvant-bound rBoNT(H(c)) protein antigens. We found that all three of the rBoNT(H(c)) proteins were susceptible to degradation via both oxidation and deamidation. In many cases, such reactions occurred earlier with the adjuvant-bound protein formulations when compared to the proteins in control samples that were not bound to adjuvant. Additionally, some chemical modifications were found in the adjuvant-bound protein formulations but were not detected in the unbound solution controls. Our studies indicate that binding to aluminum-based adjuvants can impact the chemical stability and/or the chemical degradation pathways of protein during long-term storage in aqueous suspension. Furthermore, the methods we developed should be widely useful for assessing chemical stability of adjuvant-bound recombinant protein antigens.

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.

Situacion actual de la vacuna de rotavirus / Current situation of rotavirus vaccine

En el año 1998 se aprobó la primera vacuna frente a rotavirus (Rotashield). Fue retirada un año después tras sospecharse su asociación con varios casos de invaginación intestinal. Posteriormente se han desarrollado nuevas vacunas con dos líneas de investigación fundamentales: 1) Vacunas recombinantes humano-bovinas: Rotateq, vacuna pentavalente desarrollada a partir de los serotipos G1-G4 y P1 humanos y la cepa WC3 bovina; vacuna tetravalente a partir de los serotipos G1-G4 humanos y la cepa UK bovina. 2) Vacunas atenuadas de cepas humanas: frente a las cadenas 89-12 de rotavirus, RIX4414 (Rotarix) y RV3 (Serotipo G3PA2). Las vías de investigación más novedosas están enfocadas en el desarrollo de vacunas DNA en modelos experimentales y de vacunas que utilizan como vehículo de expresión alimentos transgénicos

In 1998 was licensed the first vaccine against rotavirus (Rotashield). This vaccine was with drawn one year later because it was suspected its association with some cases of intussusception. Consequently, new vaccines have been developed in two primary ways of investigation: 1) The human-bovinere assortant attenuated vaccines: Rotateq, a WC3 bovine strain mixed with G1-G4 and P1 human serotypes pentavalent vaccine; the UK bovine strain mixed with G1-G4 human serotypes tetravalent vaccine. 2) The live, attenuated human rotavirus vaccines: against 89-12 strain, RIX4414 (Rotarix) and RV3 (G3PA2 serotype). The newest lines of investigation are focused on the development of DNA vaccines in experimental models and vaccines which are expressed in transgenic aliments

Concentration determination of a recombinant vaccine antigen adsorbed onto an alum adjuvant by chemiluminescent nitrogen detection.

PURPOSE: A chemiluminescent nitrogen detector (CLND) has been evaluated for determining the concentration of an aluminum-adsorbed recombinant vaccine antigen. METHODS: Quantification of the antigen was based upon several nitrogen-containing compounds used to calibrate the CLND. All calibrants (6.75-400 microg/ml) generated linear standard curves, with slopes being directly proportional to the % nitrogen. The limit of quantification (LOQ) was determined to be 6.75 microg/ml based on the performance of the antigen standard curve, and the limit of detection (LOD) was defined by setting the CLND minimum peak area to 40,000 U. The CLND was capable of analyzing antigen-adjuvant suspensions (adsorbed + unbound antigen) without any sample pretreatment. To measure unbound antigen, the suspension was centrifuged and an aliquot of supernatant removed for analysis; the difference between these two measurements was the amount of adsorbed antigen. RESULTS: The adjuvant exhibited no significant matrix effect. Samples were analyzed in triplicate with observed relative standard deviation values ranging from 0.065% to 10.0%. The most accurate concentrations of the antigen were recovered relative to the antigen itself and to glycine as standards. CONCLUSION: This methodology provides a direct measurement of the concentration of a vaccine antigen adsorbed onto an aluminum adjuvant.

Oral immunization of piglets with recombinant F4 fimbrial adhesin FaeG monomers induces a mucosal and systemic F4-specific immune response.

The importance of adhesins in the pathogenicity of several bacteria resulted in studies on their usefulness in vaccines. In this study, the gene of the F4(K88)-fimbrial adhesin FaeG of the pathogenic enterotoxigenic Escherichia coli (ETEC) strain GIS26 was cloned in the pET30Ek-LIC vector and expressed with an N-terminal His- and S-tag in the cytoplasm of BL21(DE3). Recombinant FaeG (rFaeG) subunits were isolated from insoluble cytoplasmic aggregates and refolded into a native-like F4 receptor (F4R)-binding conformation. Indeed, the presence of conformational epitopes was shown by ELISA and the ability to bind the F4R was observed by inhibiting the adhesion of F4+ ETEC to F4R+ villi with increasing concentrations of native-like refolded rFaeG subunits. The rFaeG subunits appear as monomers, whereas the purified F4 fimbriae are multimers. Oral immunization of newly weaned piglets with native-like rFaeG induced a mucosal and systemic F4-specific immune response, significantly reducing F4+ E. coli excretion from 2 till 5 days following challenge infection. However, improvement of stability and immunogenicity of rFaeG is necessary since a higher F4-specific response was obtained following immunization with purified F4 fimbriae. Furthermore, the N-terminal fusion of a His- and S-tag was not detrimental for binding the F4R, supporting the use of FaeG as mucosal carrier. In conclusion, oral immunization with a recombinant fimbrial adhesin subunit of Escherichia coli induces a mucosal and systemic fimbriae-specific immune response.

Enhanced expression of a recombinant malaria candidate vaccine in Escherichia coli by codon optimization.

This study was conducted to compare the expression of three constructs of a multistage candidate vaccine (FALVAC-1) against Plasmodium falciparum in an Escherichia coli system: a synthetic gene with P. falciparum codons, a synthetic gene with optimized E. coli codons, and a synthetic gene with P. falciparum codons co-transformed with a RIG plasmid, which encodes three tRNAs (AG(A/G), ATA, GGA) that recognize rare E. coli codons. The expression of the protein increased at least threefold with codon optimization. The presence of the RIG plasmid in the co-transforming cells did not significantly increase the expression level of the gene with P. falciparum codons. The growth of cells transformed by the construct with P. falciparum codons was significantly slower than that of cells transformed by the construct with optimized E. coli codons after induction of protein expression with IPTG. The cells containing the non-codon optimized gene co-expressed with RIG plasmid had the slowest growth at all time points in culture. Thus, codon optimization significantly increases the yield of P. falciparum candidate vaccines in the E. coli expression system.

Residual DNA quantification in clinical batches of BBG2Na, a recombinant subunit vaccine against human respiratory syncytial virus.

BBG2Na, a well-defined recombinant protein produced in Escherichia coli, is a promising human respiratory syncytial virus subunit vaccine candidate. This study describes the quantification of residual DNA in large scale batches used in phase I to III clinical trials. Two different analytical methods were developed and applied on five different final bulks of Drug Substance and their associated in process control samples, namely a chemiluminescent hybridisation assay and the total DNA Threshold System assay. These two complementary methods demonstrated the clearance of residual DNA during the downstream purification process. The amount of residual DNA found in the final bulks was below 20 pg of DNA per 300 microg BBG2Na, the highest tested clinical dose of antigen. This is very low level of residual DNA for a recombinant subunit vaccine produced in a bacteria and contribute to make for BBG2Na a well-characterised biopharmaceutical. This study also provides data concerning the validation of the hybridisation dot blot assay and the total DNA Threshold(trade mark)assay.

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.

Eficacia comparativa de las vías intradérmica e intramuscular en la inmunización activa contra la hepatitis B: resultados a largo plazo / Comparative efficacy of intradermally and intramuscular routes for active immunization against hepatitis B: long-term results

Se presentan los resultados a largo plazo de un estudio prospectivo, comparativo y randomizado con 119 voluntarios jóvenes sero-negativos, con riesgo aumentado de infección por el VHB, que evaluó la inmunogenicidad comparativa de una vacuna recombinante contra la hepatitis B, administrada por vía intramuscular (IM) o intradérmica (ID). La vacuna fue administrada en tres dosis consecutivas, en un esquema de inmunización de corta duración de 0,1 y 2 meses. Una dosis de refuerzo fue administrada al cabo de 1 año, por la misma vía utilizada en la inmunización original. La distribución por edad y sexo en ambos grupos fue similar. Los resultados obtenidos al cabo de las tres dosis mostraron tasas de seroconversión y seroprotección similares para ambos grupos (96 por ciento y 96 por ciento para la vía IM Vs. 98 por ciento y 93 por ciento para la vía ID, respectivamente). El promedio geométrico del título de anti-HBsAg en el suero de los individuos inmunizados por vía IM (155 UI/L) fue significativamente superior al obtenido en aquellos inmunizados por vía ID (71 UI/L). Por otro lado, la tasa de buenos respondedores (niveles de anti-HBsAg >100 UI/L ) fue igualmente más elevada en los vacunados por vía IM (67 por ciento Vs. 39 por ciento). El porcentaje de buenos respondedores en las mujeres fue consistentemente superior al de los varones para ambos grupos (75 por ciento Vs. 55 por ciento vía IM y 54 por ciento Vs. 13 por ciento vía ID, respectivamente). En 82 individuos estudiados 1 años más tarde, el promedio geométrico de los niveles de anti-HBsAg fue significativamente superior en los que recibieron la vía IM (278 UI/L) en comparación con los vacunados por vía ID (82,3 UI/L) y la tasa de seroprotección fue también superior (100 por ciento Vs. 84,8 por ciento respectivamente). De la misma manera, la respuesta de anti-HBsAg sérica a la dosis de refuerzo, fue mejor en los vacunados por vía IM (promedio geométrico 5.090 UI/L) con respecto a los que recibieron la vía ID.