Oral rotavirus vaccine shedding as a marker of mucosal immunity.

Group A rotaviruses (RVA) remain a leading cause of pediatric diarrhea worldwide, in part due to underperformance of currently approved live-attenuated, oral vaccines in low-and-middle income countries. Improved immune correlates of protection (CoP) for existing oral vaccines and novel strategies to evaluate the performance of next-generation vaccines are needed. Use of oral vaccines as challenge agents in controlled human infection models is a potential approach to CoP discovery that remains underexplored. In a live-attenuated, oral rotavirus vaccine (Rotarix, GlaxoSmithKline) efficacy trial conducted among infants in Dhaka, Bangladesh, we explored the potential for the second dose of the two-dose series to be considered a challenge agent through which RVA immunity could be explored, using fecal virus shedding post-dose 2 as a marker of mucosal immunity. Among 180 vaccinated infants who completed the parent study per protocol, the absence of fecal vaccine shedding following the second dose of Rotarix suggested intestinal mucosal immunity generated by the first dose and a decreased risk of RVA diarrhea through 2 years of life (RR 0.616, 95% CI 0.392-0.968). Further development of controlled human infection models for group A rotaviruses, especially in prospective studies with larger sample sizes, may be a promising tool to assess rotavirus vaccine efficacy and CoPs.

Pathogenic and Transmission Potential of Wildtype and Chicken Embryo Origin (CEO) Vaccine Revertant Infectious Laryngotracheitis Virus.

Infectious laryngotracheitis (ILT) is an infectious upper respiratory tract disease that impacts the poultry industry worldwide. ILT is caused by an alphaherpesvirus commonly referred to as infectious laryngotracheitis virus (ILTV). Vaccination with live attenuated vaccines is practiced regularly for the control of ILT. However, extensive and improper use of live attenuated vaccines is related to vaccine viruses reverting to virulence. An increase in mortality and pathogenicity has been attributed to these vaccine revertant viruses. Recent studies characterized Canadian ILTV strains originating from ILT outbreaks as related to live attenuated vaccine virus revertants. However, information is scarce on the pathogenicity and transmission potential of these Canadian isolates. Hence, in this study, the pathogenicity and transmission potential of two wildtype ILTVs and a chicken embryo origin (CEO) vaccine revertant ILTV of Canadian origin were evaluated. To this end, 3-week-old specific pathogen-free chickens were experimentally infected with each of the ILTV isolates and compared to uninfected controls. Additionally, naïve chickens were exposed to the experimentally infected chickens to mimic naturally occurring infection. Pathogenicity of each of these ILTV isolates was evaluated by the severity of clinical signs, weight loss, mortality, and lesions observed at the necropsy. The transmission potential was evaluated by quantification of ILTV genome loads in oropharyngeal and cloacal swabs and tissue samples of the experimentally infected and contact-exposed chickens, as well as in the capacity to produce ILT in contact-exposed chickens. We observed that the CEO vaccine revertant ILTV isolate induced severe disease in comparison to the two wildtype ILTV isolates used in this study. According to ILTV genome load data, CEO vaccine revertant ILTV isolate was successfully transmitted to naïve contact-exposed chickens in comparison to the tested wildtype ILTV isolates. Overall, the Canadian origin CEO vaccine revertant ILTV isolate possesses higher virulence, and dissemination potential, when compared to the wildtype ILTV isolates used in this study. These findings have serious implications in ILT control in chickens.

Multiple recombination events between field and vaccine strains resulted in the emergence of a novel infectious bronchitis virus with decreased pathogenicity and altered replication capacity.

In this study, we isolated and identified 2 infectious bronchitis virus (IBV) strains from layer chickens soon after vaccination with the Massachusetts-Connecticut bivalent vaccine (Conn) and H120 and 4/91 booster vaccines in China in 2011. The results of cross-virus-neutralization tests and phylogenetic analysis of the S1 subunit of spike gene of these vaccine strains and other reference strains showed that strain LJL/110302 was of GI-19 lineage, whereas LLN/111169 was of the GI-1 lineage of the Conn serotype. Further comparative genomic analysis revealed that LLN/111169, an IBV strain with novel traits, originated from multiple recombination events (at least 3 recombination sites) between GI-19 and the Conn and 4/91 vaccine strains. LLN/111169 was pathogenic to specific pathogen-free (SPF) chickens. This is of prime importance because while IBV prevention measures worldwide are mainly dependent on modified live vaccine strains, our results showed that recombination between field and vaccine strains has produced a novel pathogenic IBV strain. In addition, LLN/111169 showed relatively broad tissue tropism (trachea, lungs, kidneys, and cecal tonsils) in infected SPF chickens. These results emphasize the importance of IBV surveillance in chicken flocks.

VP7 and VP4 genotypes of rotaviruses cocirculating in Iran, 2015 to 2017: Comparison with cogent sequences of Rotarix and RotaTeq vaccine strains before their use for universal mass vaccination.

The present study was conducted to analyze the genotypic diversity of circulating species A rotavirus (RVA) strains in Iran and also to investigate comparative analysis between the genotypes of VP4 and VP7 of cocirculating RVA and vaccine strains before the vaccine is introduced in the national immunization program. The G3-lineage I was found in this study as the most common G genotype which was followed by G9-lineage III, G1-lineages I, II, G12-lineage III, G2-lineage IV, and G4-lineage I. Also, P[8]-lineages III, IV was found as the predominant P genotype which was followed by P[4]-lineage V, and P[6]-lineage I. Overally, G3P[8] was determined as the most common combination. Moreover, the analysis of the VP7 antigenic epitopes showed that several amino acid differences existed between circulating Iranian and the vaccine strains. The comparison of genotype G1 of Iranian and vaccine strains (RotaTeq and Rotarix), and genotypes G2, G3, and G4 of Iranian and RotaTeq vaccine strains revealed three to five amino acids differences on the VP7 antigenic epitopes. Furthermore, analyzing of the VP8* epitopes of Iranian P[8] strains indicated that they contained up to 11 and 14 amino acid differences with Rotarix and RotaTeq, respectively. Based on different patterns of amino acid substitutions in circulating and vaccine strains, the emergence of antibody escaping mutants and potentially the decrease of immune protection might ensue in vaccinated children. However, considering the broad cross-protective activity of RVA vaccines, their efficacy should be monitored after the introduction in Iran.

A New Strategy for the Detection of Chicken Infectious Anemia Virus Contamination in Attenuated Live Vaccine by Droplet Digital PCR.

Chicken infectious anemia virus (CIAV) causes the atrophy of bone marrow hematopoietic and lymphoid tissues in chicks, leading to huge economic losses all over the world. The using of attenuated vaccine contaminated with CIAV increased the mortality and the pathogenicity of other diseases in many farms. However, it is difficult to detect the CIAV contamination by general detection technology due to the extremely low dose of CIAV in vaccines. In this study, we established a new method called droplet digital Polymerase Chain Reaction (ddPCR) to detect CIAV contamination of vaccines more sensitively and accurately. The lowest detection limitation of this method is 2.4 copies of CIAV plasmid or CIAV contamination at 0.1 EID50/1000 feathers in vaccines without any positive signals of other viruses. Besides, the sensitivity of ddPCR is 100 times greater than that of conventional PCR and 10 times greater than that of real-time PCR. The ddPCR technique is more sensitive and more intuitive. Therefore, it could be valuable for the detection of CIAV contamination in vaccines.

Development of a live attenuated trivalent porcine rotavirus A vaccine against disease caused by recent strains most prevalent in South Korea.

Porcine rotaviruses cause severe economic losses in the Korean swine industry due to G- and P-genotype mismatches between the predominant field and vaccine strains. Here, we developed a live attenuated trivalent porcine group A rotavirus vaccine using 80 cell culture passages of the representative Korean predominant strains G8P[7] 174-1, G9P[23] PRG942, and G5P[7] K71. Vaccination with the trivalent vaccine or its individual components induced no diarrhea during the first 2 weeks post-vaccination, i.e., the vaccines were attenuated. Challenge of trivalent-vaccinated or component-vaccinated piglets with homologous virulent strain(s) did not induce diarrhea for 2 weeks post-challenge. Immunization with the trivalent vaccine or its individual components also alleviated the histopathological lesions in the small intestines caused by challenge with the corresponding original virulent strain(s). Fecal secretory IgAs specific for each of vaccine strains were detected starting at 14 days post-vaccination (dpv), and IgA levels gradually increased up to 28 dpv. Oral immunization with the trivalent vaccine or its individual components induced high levels of serum virus-neutralizing antibody by 7 dpv. No diarrhea was observed in any experimental piglets during five consecutive passages of each vaccine strain. Our data indicated that the live attenuated trivalent vaccine was safe and effective at protecting piglets from diarrhea induced by challenge exposure of homologous virulent strains. This trivalent vaccine will potentially contribute toward controlling porcine rotavirus disease in South Korea and other countries where rotavirus infections with similar G and P genotypes are problematic.

Synergetic pathogenicity of Newcastle disease vaccines LaSota strain and contaminated chicken infectious anemia virus.

Newcastle disease virus (NDV)-attenuated vaccine has been widely used to prevent ND in poultry flocks, while many reports also mentioned the exogenous virus contamination in attenuated vaccines, which might be the reason for the widespread of some contagious diseases. Recently, the chicken infectious anemia virus (CIAV) contamination in the NDV-attenuated vaccine was also found in China, though no systemic study has studied the pathogenicity or infection mechanism of this special transmission route. Accordingly, simulation experiments were launched using CIAV isolated from a contaminated NDV-attenuated vaccine. Results showed that using NDV-attenuated vaccine contaminated with CIAV could cause CIA in chickens with obvious symptoms, including anemia, hemorrhage, lymphoatrophy, and growth retardation, while the synergistic reaction of CIAV and LaSota prompted their multiplication in vivo and disturbed the production of antibodies against each other. And CIAV could significantly reduce the NDV antibody titers and decrease the protective effectiveness. This study showed the synergetic pathogenicity of CIAV and LaSota strain after using contaminated NDV-attenuated vaccine, helping us to understand how the CIAV causes infection and induces severe diseases with a relatively low dose through the mouth, as well as reminding us that the damage of an attenuated vaccine contaminated with CIAV even in extremely low dose is not insignificant.

Development of Pyrosequencing Assay for Evaluation of Genetic Stability of Vaccine Strains of Live Attenuated Influenza Type B Vaccine.

We developed a pyrosequencing protocol for monitoring of stability of attenuating mutations in the genome of vaccine reassortants based on master donor virus of Russian live attenuated influenza vaccine B/USSR/60/69. The developed protocol allows rapid and accurate assessment of mutations and can be used for analysis of genetic stability of reassortants during vaccine strain development and manufacturing, as well as genetic stability of vaccine isolates of influenza B virus during pre-clinical and clinical trials.

Porcine circovirus type 1 was undetected in vaccine but could be cultured in the cell substrate of Lanzhou lamb rotavirus vaccine.

In 2010, Rotarix was found to be contaminated with infectious porcine circovirus type 1 (PCV1). In China, the Lanzhou lamb rotavirus (LLR) vaccine is the only vaccine used to prevent rotavirus disease. From 2006 to September 2014, more than 54 million doses of LLR vaccines have been lot released. It is a safety issue whether PCV1 is present in the LLR vaccine. Although the cell substrate of LLR, bovine kidney (BK), is different from that of Rotarix, we have investigated the cell's permissivity for PCV1 by both infectivity and full-length PCR analysis. We have assessed the LLR using a quantitative PCR (qPCR) assay. A total of 171 random batches of LLR final products over a period of 5 years were tested, and no PCV1 was detected (0/171). Infectivity studies showed that two strains of PCV1, the PCV1-prototype, which was derived from PK-15 cells, and the mutant, PCV1-GSK, which was isolated from Rotarix, were capable of replicating in BK cells over a wide m.o.i. ranging from 10 to 0.01. After culture for 6 days, copies of PCV1-prototype DNA were higher than those of PCV1-GSK on average. The genome of the virus was detected at 6 days post-infection. In summary, the LLR vaccine is free of PCV1. Nevertheless, because PCV1 can replicate in the BK cell substrate, manufacturers need to be vigilant in monitoring for this adventitious agent.

Salmonella Gallinarum field isolates and its relationship to vaccine strain SG9R.

1. The aim of the present study was to determine if the 9R-strain of the Salmonella Gallinarum live vaccine was responsible for having fowl typhoid outbreaks in chicken flocks from both chicken and turkey breeders as well as to verify the antimicrobial resistance of the isolates from the outbreaks. 2. The triplex polymerase chain reaction, standard antimicrobial test, beta-lactamase genes identification and Ion Torrent PMG whole-genome sequence were used in the field isolates and in the vaccine strain of S. Gallinarum. 3. The 60 tested isolates were not from vaccine origin and manifested high resistance to drugs from macrolide and quinolone groups. Whole-genome sequencing (WGS) and single nucleotide polymorphism analysis on selected isolates for core genes from Salmonella enterica confirmed the wild origin of these isolates and showed two possible sources of S. Gallinarum in the studied outbreaks. 4. S. Gallinarum isolated from fowl typhoid outbreaks in the studied period were not caused by the use of the SG9R live vaccine. The source of strains sequenced was diverse.

Isolation, identification, and hexon gene characterization of fowl adenoviruses from a contaminated live Newcastle disease virus vaccine.

To investigate the possible causes of the massive spread of fowl adenovirus (FAdV) infection among chickens in recent years in China, 32 batches of live-virus vaccines were tested for contamination with FAdV by PCR. Among these, 1 live Newcastle disease virus (NDV) vaccine of the LaSota strain was demonstrated to be positive for contamination. The amplified hexon gene exhibited 99.8% identity with a recent Chinese field isolate (JSJ13) of FAdV-4. The positive LaSota vaccine was first neutralized with anti-NDV serum and then inoculated into specific pathogen-free embryos at embryonic day 5 through the yolk sac for isolation of the contaminated FAdV. The same hexon gene bands were amplified from extracted DNA of the liver tissues and chicken embryo allantoic fluid of the inoculated embryos, indicating the replication and isolation of the FAdV-4 virus strain that had contaminated the vaccine. This represents the first report of FAdV-4 contamination in a live vaccine for poultry in China. These findings suggest that contamination of live vaccine might represent one of the most important causes of massive outbreaks of FAdV infection among chickens and indicate that FAdV should therefore be included in the regular monitoring list for the detection of exogenous viral contamination of attenuated vaccines for poultry.

Development of a real-time quantitative RT-PCR to detect REV contamination in live vaccine.

Based on the published Avian reticuloendotheliosis virus (REV) whole genome sequence, primers and TaqMan probes were designed and synthesized, and the TaqMan probe fluorescence real-time quantitative RT-PCR (qRT-PCR) method for detecting the REV pol gene was established by optimizing the reaction conditions. Sensitivity analysis showed that the qRT-PCR method had a sensitivity that was 1,000-fold higher than conventional PCR. Additionally, no amplification signals were obtained when we attempted to detect DNA or cDNA of ALV-A/B/J, MDV, CIAV, IBDV, ARV, NDV, AIV, or other viruses, suggesting a high specificity for our method. Various titers of REV were artificially "spiked" into the FPV and MDV vaccines to simulate REV contamination in attenuated vaccines to validate this qRT-PCR method. Our findings indicated that this qRT-PCR method could detect REV contamination at a dose of 1 TCID50/1,000 feathers, which was 10,000-fold more sensitive than the regular RT-PCR detection (10(4) TCID50/1000 feathers).

Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry.

Infection by dengue flavivirus is transmitted by mosquitoes and affects tens to hundreds of millions people around the world each year. Four serotypes have been described, all of which cause similar disease. Currently, there no approved vaccines or specific therapeutics for dengue, although several vaccine prototypes are in different stages of clinical development. Among them, a chimeric vaccine, built from the replication machinery of the yellow fever 17D virus, has shown promising results in phase III trials. Accurate quantitation of expressed viral particles in alive attenuated viral antigen vaccine is essential and determination of infectious titer is usually the method of choice. The current paper describes an alternative or orthogonal strategy, namely, a multiplexed and absolute assay of four proteins of the chimera yellow fever/dengue serotype 4 virus using targeted MS in SRM mode. Over 1 month, variability of the assay using a partially purified Vero cell extract was between 8 and 17%, and accuracy was between 80 and 120%. In addition, the assay was linear between 6.25 and 200 nmol/L and could therefore be used in the near future to quantify dengue virus type 4 during production and purification from Vero cells.

Development and validation of an egg-based potency assay for a trivalent live attenuated influenza vaccine.

Live attenuated influenza vaccines (LAIVs) targeting seasonal influenza are produced in embryonated eggs and formulated as a trivalent preparation of three live attenuated vaccine strains, one A(H1N1) strain, one A(H3N2) strain and one type B strain. In this study, we describe an egg-based potency assay for estimating the 50% Egg infectious dose (EID50) of individual strains in the trivalent preparation by selective neutralisation of two strains and then estimating the infective titres of the non-neutralised strain. The test is highly specific, and no cross interference of heterologous antisera is observed in the estimation of individual titres. Individual strains with titres in the range of 6.5-7.0 log EID50 per 0.5 ml show intra-assay and inter-assay coefficients of variance ranging from 1.25% to 2.95%. This assay was developed to establish a simple, reliable and inexpensive egg-based assay for estimating the potency of individual strains in a trivalent preparation.

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.

Influenza: profilaxis mediante la inmunización activa / Influenza: prophylaxis through active immunization

Las alternativas médicas disponibles para enfrentar las infecciones por el virus influenza se han optimizado por el desarrollo de vacunas más seguras y mejor toleradas que las alternativas de inmunización originales. La eficacia de la inmunización activa para este virus ha sido demostrada en varios ensayos clínicos que han indicado un claro beneficio para la población vacunada y que alcanza eficacias superiores a 70 por ciento en la población general y al menos 50 por ciento en los pacientes sobre 65 años de edad. Las formulaciones parenterales constituyen actualmente la alternativa predominante en el mercado (ya sea con virus totales, fragmentados o con antígenos purificados) y pueden ser aplicadas universalmente, incluso en pacientes inmunosuprimidos, porque contienen virus inactivados. Las alternativas con virus atenuados están en desarrollo, no están aún disponibles y podrían estar contraindicadas en algunos pacientes inmunosuprimidos. Su ventaja radica en su aplicación nasal que podría facilitar su adherencia en las campañas de inmunización

Detection of wild-type Aujeszky's disease virus by polymerase chain reaction in sheep vaccinated with a modified live vaccine strain.

An outbreak of Aujeszky's disease occurred in a flock of sheep which had been housed together with pigs. After the death of five sheep with clinical signs of Aujeszky's disease, the remaining sheep were vaccinated with the Bartha vaccine strain, and the pigs were vaccinated with the 783 vaccine strain of Aujeszky's disease virus. Despite vaccination, however, more sheep died. Brain tissues from four sheep were collected for virus isolation and for immunohistological examinations. Only vaccine virus (gE-negative) was detected in the tissue. After DNA restriction enzyme analysis of the isolated virus, DNA of one or both of the vaccine strains was detected in all sheep. In one sheep field virus DNA was also detected. However, when the polymerase chain reaction was performed on samples prepared from paraffin-embedded tissues, DNA of field virus (gE-positive) was detected in all four sheep. It was probable that the sheep had not yet mounted a sufficient immune response to the vaccine virus, or were already infected with field virus at the time of vaccination. We concluded that the sheep died from field virus infection and not from vaccine virus infection and that only the polymerase chain reaction made it possible to specifically detect even very small amounts of field virus DNA among vaccine virus DNA in all investigated sheep.

Laboratory tests for live attenuated poliovirus vaccines.

A new generation of tests to control live attenuated poliovirus vaccines are under development based on major advances in our understanding of the molecular basis of attenuation and reversion to virulence of polioviruses. These include an alternative in vivo neurovirulence test in transgenic mice that express the human poliovirus receptor and a new in vitro test, the MAPREC (mutant analysis by polymerose chain reaction and restriction enzyme cleavage assay, that assesses consistency of production at a molecular level. Excellent progress is being made with both methods but neither is sufficiently developed yet for regulatory use. Critical review of existing control tests shows that the WHO neurovirulence test is well standardized and contributes significantly to the assessment of each batch. On the other hand, the current rct40 test is neither standardized nor particularly informative, though improvements could be made in both areas. The continued relevance of other marker tests such as the d or antigenic marker is doubtful. Potency, identity and thermal stability tests are crucial for control of the final trivalent vaccine.