A live attenuated turkey rhinotracheitis virus vaccine. 2. The use of the attenuated strain as an experimental vaccine

An attenuated turkey rhinotracheitis (TRT) vaccine strain, when given either by eye drop or by aerosol, has been shown to be effective in protecting 1- to 11-day-old turkey poults against an experimental challenge with TRTV up to 14 weeks later. TRT antibody-free poults and poults hatched from TRT-immune dams were both equally well protected, although a high proportion of the latter did not respond serologically to the vaccine. As little as 1 CD50 of vaccine was found to be effective in protecting poults against challenge. Immunity developed within about 6 days of vaccination of 7-day-old TRT antibody-free poults and humoral antibody persisted for at least 14 weeks after a single vaccination at 4 weeks of age.     

Development of a live attenuated vaccine against turkey rhinotracheitis

Three preparations of a strain of turkey rhinotracheitis (TRT) virus were tested for their ability to protect turkey poults against challenge with virulent virus given 3 weeks later. The preparations were as follows: one had been passaged in turkey embryo tracheal organ culture (TOC) 98 times, another had been passaged in primary chick embryo fibroblast (CEF) monolayers 28 times and the third had undergone 17 passages in Vero cell monolayers. Each was administered by the eyedrop route to groups of 21-day-old TRT-seronegative turkey poults. The TOC preparation caused clinical signs consistent with TRT infection, indicating the virus had not been attenuated. The CEF and Vero preparations produced no clinical effects. Following challenge with virulent TRT virus at 21 days post-inoculation, the CEF group developed clinical signs consistent with TRT but the TOC and Vero virus groups showed none. All other parameters correlated with these findings. All groups showed an increase in specific SN and ELISA antibodies following challenge. These results indicated that after relatively few passages in Vero cells, this strain of TRT virus became satisfactorily attenuated and was able to offer protection against clinical disease following experimental challenge. Two of the three virus preparations (TOC and Vero) were also shown to spread from the inoculated birds to uninoculated contact birds, introduced into the groups at 5 days post-inoculation, and they induced protection in these contacts against virulent virus challenge.     

Further studies on the development of a live attenuated vaccine against turkey rhinotracheitis

Turkey rhinotracheitis (TRT) virus attenuated by passaging in Vero cells was tested at two different passage levels (15 or 25 passages) and two dose levels [10(3) or 10(4) TCID50 (50% tissue culture infectious doses) per bird] to determine the efficacy in protecting turkey poults against experimental challenge with virulent TRT virus. Following administration by the eyedrop route at 10 days of age, all four preparations proved successful in providing protection against clinical disease and establishment of challenge virus in the trachea when challenged with virulent virus 3 weeks later. Twelve-day-old poults given the 25th Vero passage TRT virus at a dose of 10(3.5) TCID50 per bird were protected against experimental challenge with virulent virus for at least 22 weeks post-primary inoculation. The 25th passage virus was tested for safety by administering ten times the dose (10(4.5) TCID50 per bird) used in the previous trial to a group of 10-day-old turkey poults. None of the birds showed any clinical signs during 21 days post-inoculation. Attempts to back-passage the virus from bird to bird were unsuccessful.     

A live attenuated West Nile virus strain as a potential veterinary vaccine

This article reviews the development of two attenuated West Nile virus (WNV) variants, WNI-25 and WNI-25A. These variants have lost the neuroinvasion trait of the parental virus. Attenuation was achieved through serial passages in mosquito cells and neutralization escape from WNV-specific monoclonal antibody. Genetic analysis reveals amino acid changes between the parental and each of the variants. The attenuated variants preserve the ability to replicate in mice and geese and to induce a protective immune response. WNI-25A was found to be a genetically stable virus. This variant was successfully used as a live vaccine to protect geese against a wild-type virulent WNV field isolate that closely resembles the WNV isolated during the 1999 New York epidemic.     

Current status of live attenuated influenza virus vaccine in the US

The efficacy and effectiveness of cold adapted live attenuated (CAIV-T, FluMist intranasal influenza vaccine is reviewed. CAIV-T consists of approximately 10(7) TCID50 per dose of each influenza A/H1N1, influenza A/H3N2, and influenza B vaccine strain. The exact strains are updated each year to antigenically match the antigens recommended by national health authorities for inclusion in the vaccine. In one year in which the vaccine strain did not well match the epidemic strain, the live attenuated vaccine induced a broad immune response that cross-reacted significantly with the drifted strain. The efficacy of CAIV-T in adults was demonstrated with challenge studies and the effectiveness of the vaccine for reducing febrile upper respiratory illness, days of missed work, and days of antibiotic use was demonstrated in a large field trial. In young children, protective efficacy against culture confirmed influenza was demonstrated in a field trial with overall protective efficacy of 92% during a two year study. Vaccine was also highly protective against a strain not contained in the vaccine, with 86% protective efficacy demonstrated against this significantly drifted virus. Effectiveness measures, including protection against febrile otitis media and visits to the doctor were demonstrated. Live attenuated vaccine provides a significant new tool to help prevent influenza.     

Efficacy and safety of an attenuated live QX-like infectious bronchitis virus strain as a vaccine for chickens

The attenuation of infectious bronchitis (IB) QX-like virus strain L1148 is described. The virus was passaged multiple times in embryonated specific pathogen free (SPF) chicken eggs, and at different passage levels samples were tested for safety for the respiratory tract and kidneys in 1-day-old SPF chickens. There was a clear decrease in pathogenicity for the respiratory tract and kidneys when the virus had undergone a large number of passages. Passage level 80 was investigated for safety for the reproductive tract in 1-day-old and 7-day-old SPF chickens. In 1-day-old chickens, 12.5% of the vaccinated birds had macroscopic lesions. No lesions were observed if the chickens had been vaccinated at 7 days of age. Passage level 80 was investigated for its ability to spread from vaccinated to non-vaccinated chickens and for dissemination in the body. The virus was able to spread from vaccinated chickens to groups of non-vaccinated chickens, and in the vaccinated birds the virus was found frequently in oro-pharyngeal and cloacal swabs. A fragment of the hypervariable region of the S1 protein of passage level 80 was sequenced and revealed nucleotide changes resulting in two amino acid substitutions. Passage level 80 was given additional passages to levels 82 and 85. Both passage levels were tested for efficacy in SPF chickens and passage level 85 was tested for efficacy in commercial chickens with maternally derived antibodies (MDA) against a challenge with QX-like strain IB D388. In both SPF chickens and chickens with MDA, the vaccines based on strain IB L1148 were efficacious against challenge.     

Development of a live attenuated dengue virus vaccine using reverse genetics

There are four serotypes of dengue (DEN1-DEN4) virus that are endemic in most areas of Southeast Asia, Central and South America, and other subtropical regions. The number of cases of severe disease associated with DEN virus infection is growing because of the continued spread of the mosquito vector, Aedes aegypti, which transmits the virus to humans. Infection with DEN virus can result in an asymptomatic infection, a febrile illness called dengue fever (DF), and the very severe disease called dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Currently, a licensed vaccine is not available. However, a tetravalent vaccine is urgently needed to prevent DF and DHF/DSS, the latter of which occurs predominantly in partially immune individuals. A live attenuated, tetravalent DEN virus vaccine candidate has been generated using reverse genetics that is able to provide immunity to each of the four serotypes of DEN. Attenuation has been achieved by generating recombinant DEN (rDEN) viruses which are modified by deletion or, alternatively, by antigenic chimerization between two related DEN viruses using the following two strategies: 1) introduction of an attenuating 30 nucleotide deletion (Delta30) mutation into the 3' untranslated region of DEN1 and DEN4; and 2) replacement of structural proteins of the attenuated rDEN4Delta30 vaccine candidate with those from DEN2 or DEN3. Attenuation of the four monovalent vaccine candidates has been achieved for rhesus monkeys or humans and an immunogenic tetravalent vaccine candidate has been formulated. The level of attenuation of each dengue vaccine component can be increased, if needed, by introduction of additional attenuating mutations that have been well characterized.     

A chimeric live attenuated vaccine against Japanese encephalitis

Japanese encephalitis is a disease of the CNS, endemic in Asia and Oceania. The disease is refractory to drug treatments and whilst the rural economies remain heavily dependent on agriculture, conditions for propagation of the disease will persist. Thus, there is a need for effective vaccines. Although some currently exist, they have their shortcomings. ChimeriVax-JE (Acambis Inc.) is a chimeric, live attenuated vaccine which expresses protective Japanese encephalitis antigens and to date has proven to be safe, effective and well-tolerated in clinical trials. It therefore appears to be a cost-effective prophylactic vaccine against this debilitating disease.     

The development of live attenuated cold-adapted influenza virus vaccine for humans

A procedure to attenuate live influenza virus of type A and type B was developed using adaptation of the virus to grow at 25 degrees C (cold adaptation; ca). Through a series of stepwise passages, two stable mutants were obtained and designated as 'Master' strains, one for type A influenza virus (A/Ann Arbor/6/60-H2N2) and one for type B influenza virus (B/Ann Arbor/1/66). These mutants were used in genetic reassortment using either the classical method or more recently described reverse genetics to update the relevant surface antigens of the circulating strains of influenza virus. The derivation is based on the concept of 6/2 where 6 signifies the six internal genes of the master strain and 2 refers to the two genes coding for the two surface glycoproteins HA and NA of the circulating influenza virus. The advantages of this vaccine were demonstrated to be (1) proper level of attenuation, (2) non-transmissibility, (3) genetic stability, (4) presence of the ca and ts markers and (5) immunogenicity involving both local and the cell-mediated immune responses. The clinical trials in infants, children, adults and elderly have provided the necessary data for eventual licensing of this vaccine. The ease of administration (intranasal) safety and high efficacy make this vaccine suitable to prevent influenza virus infection in all age groups.     

Conditional live virus as a novel approach towards a safe live attenuated HIV vaccine

To control the worldwide spread of HIV, a safe and effective prophylactic vaccine is urgently needed. Studies with the simian immunodeficiency virus demonstrated that a live attenuated virus can be effective as a vaccine, but serious concerns about the safety of such a vaccine virus have arisen. We propose a conditional live virus, of which the replication can be switched on and off at will, as a novel approach for an HIV vaccine.     

Response of seronegative and seropositive adult volunteers to live attenuated cold-adapted reassortant influenza A virus vaccine.

The infectivity and immunogenicity of live attenuated A/Washington/897/80 cold-adapted reassortant virus vaccine was evaluated in seronegative (hemagglutination inhibition titer, less than or equal to 1:4) and seropositive (hemagglutination inhibition titer, greater than 1:4) adult volunteers. The vaccine was efficient in infecting seronegative volunteers (94%). Moreover, 51% of seropositive vaccinees were infected by the virus. After live virus vaccination, greater than 83% of both seronegative and seropositive vaccinees achieved a level of nasal wash antibody previously associated with resistance to infection with influenza A virus. These findings indicate that both seronegative and seropositive vaccinees can benefit from live virus vaccination.     

Cold-adapted, live attenuated influenza vaccine

The recently licensed cold-adapted, live attenuated influenza vaccine (CAIV-T, FluMist, MedImmune Vaccines Inc.) has the potential to enhance control of epidemic influenza. The intranasal vaccine has proven safety and efficacy. Regulatory constraints and cost of CAIV-T have hampered the introduction of the vaccine in the first year. Unwarranted concern about possible transmission of the virus from vaccine recipients to immunocompromised patients limited use in healthcare personnel. The intense influenza A(H3N2) epidemic of 2003-2004 has underscored the necessity of supplementing current efforts to control influenza. Over 120 deaths have been documented in children - the majority of which have been previously healthy. Use of CAIV-T in children will not only decrease the risk of serious disease, but also dampen the spread of the virus in the community and reduce exposure of patients who are at high risk of complications and death.     

RT-PCR-ELISA方法检测甲肝减毒活疫苗病毒滴度的初步研究

目的 应用核酸扩增产物测定的固相杂交酶联显色法(RT-PCR-ELISA)检测甲肝减毒活疫苗病毒滴度。方法 应用RT-PCR-ELISA。将标记有生物素的寡核苷酸引物所扩增的疫苗病毒基因产物。与微孔反应板上的特异性探针进行快速杂交，通过辣根过氧化物酶标记的链亲和素进行酶联显色。读取吸光度(A值)。判断结果。应用此法检测了11批甲肝活疫苗滴度。并与常规细胞培养法(CCID50)比较。结果 本方法与细胞培养法的敏感性相仿，具有简便、快速、特异的优点。结论 RTPCR-ELISA法有望代替常规细胞培养法应用于甲肝减毒活疫苗病毒滴度的检测。     

Evaluation of a live attenuated, cold-adapted parainfluenza virus type 3 vaccine in children.

Cold passage 18 (CP18) parainfluenza virus type 3 (PIV-3) vaccine was evaluated in a double-blind, randomized, placebo-controlled study of 95 infants and young children. None of 19 seropositive older children 41 to 124 months old became infected when 10(6) 50% tissue culture infective doses (TCID50) of vaccine virus was administered intranasally. Two of nine and seven of twenty-four young seropositive children given 10(5) or 10(6) TCID50 of CP18 PIV-3, respectively, became infected. Each of four seronegative young children became infected, as indicated by virus shedding and antibody response, when given 10(6) TCID50 of CP18 PIV-3 intranasally. Illness was not observed in seropositive children. Two of the four seronegative children developed a mild illness characterized by rhinorrhea and wheezing on auscultation; none had fever. In one case, vaccine virus spread from a vaccine to a sibling control but did not cause illness. The vaccine is attenuated relative to wild-type PIV-3, but additional attenuation will be required to achieve a satisfactory PIV-3 vaccine.     

A comparison of three methods for detecting antibodies to turkey rhinotracheitis virus

Sera from a flock of naturally infected commercial turkeys were tested for antibodies against turkey rhinotracheitis virus using an indirect immunofluorescence test with infected turkey embryo tracheal organ cultures, a serum neutralisation test using chick embryo fibro-blasts or liver cells and an ELISA. Antibodies were detected by all tests within 5 days of the appearance of clinical respiratory disease. Serum neutralising antibodies detected in chick embryo fibroblasts rapidly achieved their peak titres and were decreasing by day 13. ELISA antibodies peaked on day 13. On days 13 and 34 significant correlations were obtained for immunofluorescence and ELISA and for ELISA and microneutralisation in fibroblasts. For both the latter tests there was a good correlation between the results obtained for individual birds within the flock on days 13 and 34. ELISA and serum neutralisation in chick embryo fibroblast monolayers would appear to be sensitive and reliable tests for detecting circulating antibodies against turkey rhinotracheitis virus in commercial flocks.     

Pneumovirus-like characteristics of the mRNA and proteins of turkey rhinotracheitis virus

Electronmicroscopy has indicated that turkey rhinotracheitis virus (TRTV), the causative agent of an acute respiratory disease in turkeys, is a member of the Paramyxoviridae family. To determine if TRTV belongs to one of the three defined genera of this family (Paramyxovirus, Morbillivirus and Pneumovirus) we have analysed the RNA and proteins induced during replication of TRTV in Vero cells. Following replication in the presence of actinomycin D 10 polyadenylated RNA bands, ranging in Mr from 0.22 to 2.0 X 10(6), were detected in infected cells; some bands probably contained 2 or more RNA species. Viral proteins were studied after radiolabelling in the presence of [35S]methionine and [3H]glucosamine. Comparison of the polypeptides in mock-infected and infected cells, virions and nucleocapsids and after lentil-lectin chromatography and immunoprecipitation revealed seven virus-specific polypeptides (p), some of which were glycosylated (gp): gp82 (Mr 82K), gp68, gp53, gp15, p43, p40 and p35. These are considered to be analogous to the large glycopolypeptide (HN, H and G), fusion protein precursor F0, the F protein cleavage products F1 and F2, nucleocapsid (N), phosphorylated (P) and matrix (M) polypeptides, respectively, of the Paramyxoviridae. Two other polypeptides (Mr 200K and 22K) were also detected, as was a glycopolypeptide of Mr 97K, probably related to gp82. Tunicamycin inhibited glycosylation of gp53 and gp15 but gp82 was little affected, most glycans still being present on a glycopolypeptide of approximately 79K. This finding, indicating that gp82 has mostly O-linked glycans, considered with the mRNA profile and the molecular weight of the N protein shows that of the three genera in this family, TRTV most closely resembles the Pneumovirus genus.     

Highly attenuated Bordetella pertussis strain BPZE1 as a potential live vehicle for delivery of heterologous vaccine candidates

Bordetella pertussis, the causative agent of whooping cough, is a promising and attractive candidate for vaccine delivery via the nasal route, provided that suitable attenuation of this pathogen has been obtained. Recently, the highly attenuated B. pertussis BPZE1 strain has been described as a potential live pertussis vaccine for humans. We investigated here the use of BPZE1 as a live vehicle for heterologous vaccine candidates. Previous studies have reported the filamentous hemagglutinin (FHA), a major B. pertussis adhesin, as a carrier to express foreign antigens in B. pertussis. In this study, we also examined the BrkA autotransporter as a surface display system. Three copies of the neutralizing peptide SP70 from enterovirus 71 (EV71) were fused to FHA or in the passenger domain of BrkA, and each chimera was expressed in BPZE1. The FHA-(SP70)₃ and BrkA-(SP70)₃ chimeras were successfully secreted and exposed at the bacterial surface, respectively. Nasal administration of the live recombinant strains triggered a strong and sustained systemic anti-SP70 antibody response in mice, although the titers and neutralizing activities against EV71 were significantly higher in the sera of mice immunized with the BrkA-(SP70)₃-producing strain. These data indicate that the highly attenuated BPZE1 strain is a potential candidate for vaccine delivery via the nasal route with the BrkA autotransporter as an alternative to FHA for the presentation of the heterologous vaccine antigens.