Aerosol Inoculation Research Articles

Ovine herpesvirus 2 replicates initially in the lung of experimentally infected sheep

Ovine herpesvirus 2 (OvHV-2), a rhadinovirus in the subfamily Gammaherpesvirinae, is the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal lymphoproliferative disease primarily of ruminants worldwide. Inability to propagate the virus in vitro has made it difficult to study OvHV-2 replication. Aerosol inoculation of sheep with OvHV-2 from nasal secretions collected from naturally infected sheep during shedding episodes results in infection of naive sheep, providing an excellent system to study OvHV-2 initial replication in the natural host. In this study, we showed that OvHV-2 delivered through the nasal route by nebulization resulted in infection in all lambs, but no infection was established in any lambs after intravenous or intraperitoneal injection. In nebulized lambs, while it was not detected initially in any other tissues, OvHV-2 DNA became detectable in the lung at 3 days post-infection (p.i.), increased to about 900 copies per 50 ng DNA at 5 days p.i., reached peak levels ( approximately 7500 copies) at 7 days p.i., and then declined to an average of 800 copies at 9 days p.i. Transcripts of OvHV-2 open reading frame 25 (coding for the capsid protein), an indicator of virus replication, were only detected in lung tissues, and were positively correlated with OvHV-2 DNA levels in the lungs. In addition, selected immune response genes were also highly expressed in the lung at 5 and 7 days p.i. The data indicate that lung is the primary replication site for OvHV-2 during initial infection in sheep and suggest that viral replication is promptly controlled by a host defence mechanism.

Laboratory methods for assessing vaccine potency retained in aerosol outputs from nebulizers: Application to World Health Organization measles aerosol project

Background Laboratory methods for measuring vaccine potency of nebulized aerosol are required to support clinical trials of measles aerosol vaccination. Methods Measles vaccine containing the Edmonston Zagreb virus strain was reconstituted in sodium fluoride as tracer and nebulized from three devices. Emitted aerosol was collected using an impinger. Aliquots were removed from the impinger chamber for vaccine virus plaque assay and for fluoride measurement to determine aerosol output. Results Vaccine potency retention results were adjusted to take into account the effect of aerosol output on estimates. Adjusted potency of nebulized vaccine ranged from 88% to 102%. Conclusions New laboratory methods to measure aerosol vaccine potency retention were reliable and accurate. The results demonstrated that Edmonston Zagreb vaccine remains robust during aerosolisation and imply that this is a viable candidate for further evaluation in the measles aerosol project.

Aerosol immunization with NYVAC and MVA vectored vaccines is safe, simple, and immunogenic

Each year, approximately five million people die worldwide from putatively vaccine-preventable mucosally transmitted diseases. With respect to mass vaccination campaigns, one strategy to cope with this formidable challenge is aerosol vaccine delivery, which offers potential safety, logistical, and cost-saving advantages over traditional vaccination routes. Additionally, aerosol vaccination may elicit pivotal mucosal immune responses that could contain or eliminate mucosally transmitted pathogens in a preventative or therapeutic vaccine context. In this current preclinical non-human primate investigation, we demonstrate the feasibility of aerosol vaccination with the recombinant poxvirus-based vaccine vectors NYVAC and MVA. Real-time in vivo scintigraphy experiments with radiolabeled, aerosol-administered NYVAC-C (Clade C, HIV-1 vaccine) and MVA-HPV vaccines revealed consistent mucosal delivery to the respiratory tract. Furthermore, aerosol delivery of the vaccines was safe, inducing no vaccine-associated pathology, in particular in the brain and lungs, and was immunogenic. Administration of a DNA-C/NYVAC-C prime/boost regime resulted in both systemic and anal-genital HIV-specific immune responses that were still detectable 5 months after immunization. Thus, aerosol vaccination with NYVAC and MVA vectored vaccines constitutes a tool for large-scale vaccine efforts against mucosally transmitted pathogens.

Comparison of the safety and protective efficacy of vaccination with glycoprotein-G-deficient infectious laryngotracheitis virus delivered via eye-drop, drinking water or aerosol

Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes respiratory disease in chickens and is commonly controlled by vaccination with conventionally attenuated virus strains. These vaccines have limitations due to residual pathogenicity and reversion to virulence. To avoid these problems and to better control disease, attention has recently turned towards developing a novel vaccine strain that lacks virulence gene(s). Glycoprotein G (gG) is a virulence factor in ILTV. A gG-deficient strain of ILTV has been shown to be less pathogenic than currently available vaccine strains following intratracheal inoculation of specific pathogen free chickens. Intratracheal inoculation of gG-deficient ILTV has also been shown to induce protection against disease following challenge with virulent virus. Intratracheal inoculation, however, is not suitable for large-scale vaccination of commercial poultry flocks. In this study, inoculation of gG-deficient ILTV via eye-drop, drinking water and aerosol were investigated. Aerosol inoculation resulted in undesirably low levels of safety and protective efficacy. Inoculation via eye-drop and drinking water was safe, and the levels of protective efficacy were comparable with intratracheal inoculation. Thus, gG-deficient ILTV appears to have potential for use in large-scale poultry vaccination programmes when administered via eye-drop or in drinking water.

Ultrastructural Changes of the Chickens Lungs after Aerosol Vaccination Against Newcastle Disease

ABSTRACTThe ultrastructural changes flowing in the lung of broiler chickens at 25 days of age after aerosol vaccination with B1 strain of Newcastle disease virus (NDV) was studied. The dynamics of the ultrastructural changes of the surfactant and cells take part in its production and metabolism were investigated on the 24th, 48th 72nd hours, 6th, 8th 15th and 21st days after the aerosol vaccination. The results of electron microscope studies confirmed that almost all components of lung connected with production and recycling of surfactant were affected by aerosol vaccination. The absence or chaotic state of surfactant, apoptotic changes of pneumocytes II type, desquamation of some of them and presence of serofibrinous exudate in most of the air capillaries on the 24th - 72nd hours after aerosol vaccination have proved that the interaction of the vaccine virus and components of the lung with attitude toward surfactant were started during the first few hours. As a result an embarrassed breading, depression, gasping, coughing, sneezing and refuse of feeding were provoked. In the next 6–8 days the changes were connected with compensation processes of surfactant production and activation of lung defense system. During 15–21 days after vaccination the lung structure was restored which demonstrate the reversible type of apoptotic changes provoked by aerosol vaccination in contrast to the cases of wild NDV infection.

Plaque reduction neutralization test for measles antibodies: Description of a standardised laboratory method for use in immunogenicity studies of aerosol vaccination

Background Clinical trials of measles vaccination administered as aerosol are planned with the aim of obtaining licensure. Measles antibody levels will be measured using the plaque reduction neutralization test (PRNT) to assess antibody responses as a surrogate marker of efficacy. Methods A working group examined laboratory protocols for measles PRNT in use at three reference centres and agreed to a standardised procedure, which was subsequently validated. Results Assay validation showed quantitative results varied approximately threefold both within and between assays. The lower limit of detection was approximately 20 milliInternational Units/mL. Conclusions A standardised laboratory protocol for measles PRNT was established and validated for use in clinical trials of aerosolized measles vaccines.

Spray drying of an attenuated live Newcastle disease vaccine virus intended for respiratory mass vaccination of poultry

A powder vaccine intended for aerosol vaccination of poultry was formulated by spray drying a live attenuated Newcastle disease virus with potential stabilizers (mannitol, trehalose, polyvinylpyrrollidone (PVP), bovine serum albumin (BSA)). Thermodynamic properties, water sorption, particle size distribution, nebulization properties, density and morphology of the powders were evaluated and the virus survival during spray drying and storage was determined by incubation in embryonated eggs and subsequent haemagglutination assay. All powders had a narrow size distribution with a median volume diameter of ±30 μm (suitable for primary respiratory vaccination of chickens) and good aerosolization characteristics. Four amorphous, hygroscopic formulations were produced (trehalose, trehalose-PVP, trehalose-BSA, trehalose-PVP-BSA), where addition of BSA was beneficial for virus survival during production and storage at 6 and 25 °C. A crystalline, non-hygroscopic powder (mannitol) had a lower stabilizing capacity during production but maintained the remaining virus titre during storage. In conclusion, the study demonstrates that it is possible to produce a dry powder formulation of an attenuated live vaccine for mass vaccination of poultry in a one-step spray drying process.

Humoral and cellular immune responses to airway immunization of mice with human papillomavirus type 16 virus-like particles and mucosal adjuvants

Cervical cancer results from cervical infection by human papillomaviruses (HPV), especially HPV16. Intramuscular administrations of HPV16 virus-like particle (VLP) vaccines have been shown to induce strong neutralizing antibody responses and protect women against genital HPV16 infection and associated lesions. However, an alternative route of administration that avoids parenteral injection might facilitate vaccine implementation, particularly in developing countries which account for the majority of the worldwide cases of cervical cancer. In addition, inducing mucosal immunity could partially overcome the substantial variation in HPV16 antibodies at the cervix seen in ovulating women. Aerosol vaccination with HPV16 VLPs was previously shown to be immunogenic in mice and in women. Here, we examine whether exposure to other respiratory viral antigens may interfere with the HPV16 VLP-specific humoral response and whether two known mucosal adjuvants, CpG oligodeoxynucleotides and a natural non-toxic Escherichia coli heat-labile enterotoxin (HLT), can enhance the immunogenicity of airway immunization (nasal or aerosol-like) of mice with HPV16 VLPs. Our data show that HLT can significantly improve anti-VLP humoral responses in serum and mucosal secretions, as well as VLP-specific proliferative responses and IFN-γ production by CD8 T cells, and that recent exposure to influenza surface antigens can diminish mucosal, but not systemic, antibody responses to the VLPs.

Aerosol and subcutaneous measles vaccine: Measles antibody responses 6 years after re-vaccination

There are no large-scale data on the long-term persistence of measles antibody after vaccination by the aerosol route. We therefore followed-up South African schoolchildren 6 years after their re-vaccination with Edmonston–Zagreb (EZ) and Schwarz (SW) measles vaccine given by aerosol and subcutaneous routes. Measles antibody levels and the proportion of children who were seropositive at year 6 remained significantly higher in the Edmonston–Zagreb aerosol group compared to the groups that received Schwarz or Edmonston–Zagreb vaccine subcutaneously.Measles re-vaccination by aerosol evokes a stronger and much longer lasting antibody response than injected vaccine and should thus provide more durable protection against measles.

Deposition of differently sized airborne microspheres in the respiratory tract of chickens

As a part of the development of an efficient dry powder aerosol vaccine for poultry, the objective of this study was to accurately determine the deposition pattern of nebulized microspheres in the airways of unanaesthetized chickens of different ages (1 day, 2 weeks and 4 weeks old). In the first part of the study, the aerosol administration method was characterized: the influence of different nebulizers and nebulizing protocols on the relative humidity in the exposure chamber, the particle size distributions, the microsphere output and single microsphere percentage were determined. In the second part, birds were exposed to nebulized fluorescently labelled polystyrene microspheres (1 to 20 microm). Respiratory and gastro-intestinal tract tissue samples were collected and the number of fluorescent microspheres per sample was determined. In 2-week-old and 4-week-old chickens, microspheres of 5 and 10 microm, respectively, were too large for deposition in the lungs and air sacs as less than 5% of these microspheres penetrated into the lower airways. The larger size of microspheres reaching the lower airways of 4-week-old birds was explained by increasing airway dimensions with age. For 1-day-old chickens, deposition in the lungs decreased from 17 to 3% with increasing particle size (1 to 20 microm), but increased in the air sacs from 6 to 20%. Consequently, the total deposition percentage in the lower airways was independent of microsphere size and even 20 microm particles were able to penetrate into the lower airways, which was attributed to mouth breathing of the 1-day-old chickens.

Measles vaccination of macaques by dry powder inhalation

Measles vaccination via the aerosol route has proven effective under field conditions, using vaccine reconstituted prior to nebulization. Inhalation of a dry powder aerosol vaccine would have additional benefits, including easier logistics of administration, reduced cold chain dependence and the potential of single dose administration. We have evaluated two candidate dry powder measles vaccine formulations in macaques. Specific immune responses were demonstrated, but levels of immunity were lower than in animals vaccinated by injection or by nebulized aerosol. These studies provide proof of principle that dry powder inhalation is a possible route for measles vaccination, but suggest that either the vaccine formulation or the method of delivery need to be improved for a better immune response.

The American cotton rat: A novel model for pulmonary tuberculosis

Several animal models are used to study Mycobacterium tuberculosis (MTB) infections, but none is a fully ideal model of human disease. The American cotton rat is an excellent model for the study of several human viral and bacterial respiratory infectious diseases, but until now has not been reported to be a model with MTB infection. Preliminary experiments were designed in which two species of cotton rats (Sigmodon hispidus and Sigmodon fulviventer) received respiratory challenges with M. tuberculosis via either intranasal or aerosol inoculation. Granulomatous disease, often with central necrosis, developed in the lungs, spleen, and lymph nodes of infected animals. The number of MTB bacilli in the lungs increased logarithmically until reaching a plateau in the second month after aerosol inoculation. There were differences in response to infection between the two species, with S. fulviventer demonstrating greater mortality than S. hispidus. Cytokine gene expression analysis by reverse transcriptase polymerase chain reaction (RT-PCR) was performed on both normal appearing and granulomatous lung tissue from infected animals. Many cytokine genes were more highly expressed in the focal areas of inflammation. Cotton rats provide another valuable tool in future research with tuberculosis.

Aerosol measles vaccination in macaques: Preclinical studies of immune responses and safety

The comparative efficacy and safety of measles vaccination via the aerosol route versus subcutaneous injection has not been fully resolved. We vaccinated cynomolgus monkeys ( Macaca fascicularis) with the live-attenuated Edmonston–Zagreb measles virus (MV) vaccine and compared different routes of administration in the immunocompetent and the immunocompromised host. Immunogenicity and protective efficacy of aerosol vaccination using devices similar to those previously used in humans were comparable to those in animals vaccinated by injection. No evidence for a safety hazard associated with the route of vaccination was detected. The results of this study support further clinical evaluation of aerosol vaccination for measles.

Early Antibody Responses to Experimental Mycobacterium bovis Infection of Cattle

Bovine tuberculosis persists as a costly zoonotic disease in numerous countries despite extensive eradication and control efforts. Sequential serum samples obtained from Mycobacterium bovis-infected cattle were evaluated for seroreactivity to mycobacterial antigens. Animals received M. bovis by aerosol, intratonsil, intranasal, or intratracheal inoculation. Assays included the multiantigen print immunoassay for determination of antigen recognition patterns, immunoblot analysis for sensitive kinetic studies, and the VetTB STAT-PAK test, a novel, rapid test based on lateral-flow technology. Responses to MPB83 were detected for all M. bovis-infected animals regardless of the route or strain of M. bovis used for inoculation. Other less commonly recognized antigens included ESAT-6, CFP-10, and MPB70. Responses to MPB83 were detectable as early as 4 weeks after inoculation, were boosted upon injection of purified protein derivatives for skin testing, and persisted throughout the course of each of the four challenge studies. MPB83-specific immunoglobulin M (IgM) was detected prior to MPB83-specific IgG detection; however, early IgM responses rapidly waned, suggesting a benefit of tests that detect both IgM- and IgG-specific antibodies. The VetTB STAT-PAK test detected responses in sera from 60% (15/25) of the animals by 7 weeks after challenge and detected responses in 96% (24/25) of the animals by 18 weeks. These findings demonstrate the potential for new-generation antibody-based tests for the early detection of M. bovis infection in cattle.

Intragranulomatous necrosis in pulmonary granulomas is not related to resistance against Mycobacterium tuberculosis infection in experimental murine models induced by aerosol.

Intragranulomatous necrosis is a primary feature in the natural history of human tuberculosis (TB). Unfortunately, this phenomenon is not usually seen in the experimental TB murine model. Artificial induction of this necrosis in pulmonary granulomas (INPG) may be achieved through aerosol inoculation of lipopolysaccharide (LPS) 3 weeks after Mycobacterium tuberculosis infection. At week 9 post-infection, the centre of primary granulomas became larger, showing eosinophilic necrosis. Interestingly, INPG induction was related to mice strains C57BL/6 and 129/Sv, but not to BALB/c and DBA/2. Furthermore, the same pattern was obtained with the induction of infection using a clinical M. tuberculosis strain (UTE 0335R) that naturally induces INPG. In all the mice strains tested, the study of pulmonary mRNA expression revealed a tendency to increase or to maintain the expression of RANTES, interferon-gamma, tumour necrosis factor and iNOS, in both LPS- and UTE 0335R-induced INPG, thus suggesting that this response must be necessary but not sufficient for inducing INPG. Our work supports that INPG induction is a local phenomenon unrelated to the resistant (C57BL/6 and BALB/c) or susceptible (129/Sv and DBA/2) background of mice strains against M. tuberculosis infection.

Progress with the development of a measles aerosol vaccine

More than one dose of measles vaccine is necessary for the sustained control of measles. The aerosol route is thought to be more immunogenic for booster doses than traditional subcutaneous injections, so we did a randomised comparative trial of aerosol and subcutaneous measles vaccines in South African schoolchildren.4327 schoolchildren (aged 5–14 years), assigned by block randomisation of classrooms, received standard titre doses of either Schwarz or Edmonston-Zagreb measles vaccines subcutaneously or by aerosol. Blood samples for antibody assay were collected before vaccination, at 1 month, and 1 year after vaccination. The main endpoints (antibody titres at 1 month and 1 year) were compared between groups.992 children had antibody titre data available for all timepoints. 14 (3·6%) of 385 children who received Edmonston-Zagreb vaccine by aerosol were seronegative 1 year after vaccination, compared with 28 (8·6%) of 326 children who received Edmonston-Zagreb subcutaneous vaccine and 39 (13·9%) of 281 children who received Schwarz subcutaneous vaccine. At 1 month, 326 (84·7%) children who received aerosol Edmonston-Zagreb vaccine had sero-converted, compared with 257 (78·8%) who received subcutaneous Edmonston-Zagreb vaccine and 176 (62·6%) who received subcutaneous Schwarz vaccine. At 1 month, only 116 (22·7%) of 511 children in the Schwarz aerosol group had seroconverted; this aerosol vaccine had no detectable potency after 2 min of nebulisation. There were no serious side-effects: about 5% of children in each group had a rash within 2 weeks of vaccination.An aerosol vaccination method that uses currently available devices and a suitably stable vaccine is effective and acceptable. This form of delivery is adaptable to mass campaigns, avoids the risks associated with injections, and could help measles eradication.

How many infective viral particles are necessary for successful mass measles immunization by aerosol?

Objectives This study characterized the performance of the “Classical Mexican Device”, which immunized 4 million children against measles, demonstrating the efficacy of aerosol vaccination. Methods Using plaque-forming units to quantify virus, the particle size distribution (Next Generation Pharmaceutical Impactor) and rate of output coupled with age specific patterns of breathing allowed the calculation of expected pulmonary deposition. Results The estimated immunization dose for infants was 30 pfu's, for small children 50, and for older children and adolescents it was 130 and 225, respectively. Conclusions These performance characteristics can be used to develop newer battery operable devices that are licensable.

Immunogenicity of aerosol measles vaccine given as the primary measles immunization to nine-month-old Mexican children

Aerosol measles vaccination has been found to be more immunogenic than subcutaneous administration as a booster in school aged children, and immunogenic in 12-month-old children as a primary dose. The objective of the study was to evaluate immunogenicity to aerosol measles vaccine in 9-month-old children. Methods Nine-months-old infants received Edmonston-Zagreb measles vaccine by aerosol (10 3.58 CCID 50/0.1 mL, estimated retained dose 10 2.81 CCID 50) or subcutaneous route (10 4.28 CCID 50/0.5 mL); cellular and humoral immunity and adverse events were assessed. Results Measles-specific T cell proliferative responses developed in 42% of children given aerosolized vaccine compared with 67% of those who received subcutaneous vaccine ( p = 0.01); the mean stimulation index (SI) was 4.4 ± 0.7 versus 6.9 ± 1, respectively, ( p = 0.05). Seroconversion rates were 33 and 92% after aerosol or subcutaneous immunization ( p < 0.001). Among infants who developed serologic responses, measles geometric mean titers (GMT; 95% CI) by neutralizing antibody assay were 215 mIU/mL (115–400) in aerosol vaccine recipients and 411 mIU/mL (345–490) in those given subcutaneous vaccine ( p = 0.06). Conclusions The proportion of 9-month-old infants who developed cellular and/or humoral immunity to measles was lower in the aerosol group but measles antibody and T cell responses were comparable among those who developed measles immunity. Differences in response rates are attributable to the lower aerosol dose. Improving aerosol delivery or increasing the dose may enhance immunogenicity of primary aerosol measles vaccination in this age group.

Conjunctival vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette-Guérin

AIM: To determine the efficacy of conjunctival vaccination of captive brushtail possums (Trichosurus vulpecula) with bacille Calmette-Guérin (BCG), as measured by immunological responses to vaccination and response to intratracheal challenge with Mycobacterium bovis. METHODS: Nine adult male brushtail possums were vaccinated by the instillation of a suspension of BCG strain Pasteur 1173P2 into the conjunctival sac of each eye. Each drop contained approximately 2.5 × 105 colony forming units (cfu). At 8 weeks post-vaccination (pv) the vaccinated possums and 10 unvaccinated possums were challenged by intratracheal instillation of approximately 100 cfu of M. bovis. Cellular immune responses to bovine purified protein derivative (PPD) antigen were measured using the lymphocyte proliferation assay (LPA). Possums surviving to 50–51 days after challenge were euth anised and subjected to detailed post-mortem examination, including histopathology, to assess protection against tuberculosis. Sections of lung and spleen were cultured for M. bovis. RESULTS: No conjunctival inflammation or other adverse reactions to the administration of the vaccine were evident macroscopically. The vaccinated group showed a systemic cellular immune response to bovine PPD antigen at 4 and 8 weeks pv, and the response at 8 weeks was significantly greater than at 4 weeks (p<0.05). Conjunctival vaccination induced significant levels of protective immunity, measured as less mass of tuberculous lesions in lung (p<0.05) and less dissemination of disease in vaccinated compared with unvaccinated possums (p<0.05). CONCLUSIONS: Conjunctival vaccination with BCG induced a significant level of protective immunity against M. bovis infection in possums. This route of vaccination, together with intranasal aerosol vaccination, could be utilised in the delivery of an aerosolised vaccine using a device that sprays the vaccine suspension into the eyes and nose of possums.

Immune responses induced by lower airway mucosal immunisation with a human papillomavirus type 16 virus-like particle vaccine

Cervical cancer results from cervical infection by human papillomaviruses (HPV), especially HPV16. Previous studies have shown that intramuscular vaccination of women with an HPV16 virus-like particle (VLP) vaccine induced a strong IgG response and protected against genital HPV16 infection. However, an alternative route of administration that avoids parenteral injection while inducing mucosal immunity might facilitate vaccine implementation in some settings, and partially overcome the substantial variation in HPV16 antibodies at the cervix seen in ovulating women. In this study, women were vaccinated with escalating doses of HPV16L1 VLPs via nasal nebulisation, bronchial aerosolisation, or a combination of intramuscular and aerosol vaccination. The alternative routes of vaccination were well tolerated and many of the volunteers who received aerosol vaccinations exhibited serum antibody titers that were comparable to those induced by intramuscular vaccination. A mucosal immune response was induced by aerosol vaccination as demonstrated by the induction of anti-HPV16 VLP IgA secreting cells in PBMC and SIgA in secretions. Our data suggest that aerosol administration of HPV VLPs may represent a potential alternative to parenteral injection.