A respiratory syncytial virus (RSV) vaccine based on parainfluenza virus 5 (PIV5)

AbstractHuman respiratory syncytial virus (RSV) is a leading cause of severe respiratory disease and hospitalizations in infants and young children. It also causes significant morbidity and mortality in elderly and immune compromised individuals. No licensed vaccine currently exists. Parainfluenza virus 5 (PIV5) is a paramyxovirus that causes no known human illness and has been used as a platform for vector-based vaccine development. To evaluate the efficacy of PIV5 as a RSV vaccine vector, we generated two recombinant PIV5 viruses – one expressing the fusion (F) protein and the other expressing the attachment glycoprotein (G) of RSV strain A2 (RSV A2). The vaccine strains were used separately for single-dose vaccinations in BALB/c mice. The results showed that both vaccines induced RSV antigen-specific antibody responses, with IgG2a/IgG1 ratios similar to those seen in wild-type RSV A2 infection. After challenging the vaccinated mice with RSV A2, histopathology of lung sections showed that the vaccines did not exacerbate lung lesions relative to RSV A2-immunized mice. Importantly, both F and G vaccines induced protective immunity. Therefore, PIV5 presents an attractive platform for vector-based vaccines against RSV infection

Use of a safe, reproducible, and rapid aerosol delivery method to study infection by Burkholderia pseudomallei and Burkholderia mallei in mice.

Burkholderia pseudomallei, the etiologic agent of melioidosis, is a saprophytic bacterium readily isolated from wet soils of countries bordering the equator. Burkholderia mallei is a host-adapted clone of B. pseudomallei that does not persist outside of its equine reservoir and causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by these organisms typically occurs via percutaneous inoculation or inhalation of aerosols, and the most common manifestation is severe pneumonia leading to fatal bacteremia. Glanders and melioidosis are difficult to diagnose and require prolonged antibiotic therapy with low success rates. There are no vaccines available to protect against either Burkholderia species, and there is concern regarding their use as biological warfare agents given that B. mallei has previously been utilized in this manner. Hence, experiments were performed to establish a mouse model of aerosol infection to study the organisms and develop countermeasures. Using a hand-held aerosolizer, BALB/c mice were inoculated intratracheally with strains B. pseudomallei 1026b and B. mallei ATCC23344 and growth of the agents in the lungs, as well as dissemination to the spleen, were examined. Mice infected with 10(2), 10(3) and 10(4) organisms were unable to control growth of B. mallei in the lungs and bacteria rapidly disseminated to the spleen. Though similar results were observed in mice inoculated with 10(3) and 10(4) B. pseudomallei cells, animals infected with 10(2) organisms controlled bacterial replication in the lungs, dissemination to the spleen, and the extent of bacteremia. Analysis of sera from mice surviving acute infection revealed that animals produced antibodies against antigens known to be targets of the immune response in humans. Taken together, these data show that small volume aerosol inoculation of mice results in acute disease, dose-dependent chronic infection, and immune responses that correlate with those seen in human infections

Bacterial loads in lungs, spleen and blood after inoculation with the MicroSprayer®.

<p>Bacteria were suspended in PBS to optical densities of 1X10⁶ (Dose 1), 1X10⁵ (Dose 2), and 1X10⁴ (Dose 3) bacteria/mL, serially diluted, and plated onto agar medium to calculate the number of viable organisms in 50 µL. The MicroSprayer® was then used to deliver 50 µL of bacterial suspensions into the lungs of mice (n=15 per dose). At the indicated time points post-inoculation, mice (n=3 per dose) were euthanized and tissues were collected, homogenized, diluted, and plated onto agar medium to determine bacterial loads. Results are expressed as the mean (± standard error) total CFU/per organ (panels A, B, C, D) and mean (± standard error) CFU/ml of blood (panels E and F). These experiments were performed on at least 2 separate occasions. <i>Bp</i>= <i>B. pseudomallei</i> 1026b, <i>Bm</i>=<i>B. mallei</i> ATCC23344. Both organisms were first detected in the spleen at 24-hr post-challenge (panels C and D). <i>Bp</i> disseminated to the organ in significantly greater numbers than <i>Bm</i> at the challenge doses of 10 and 1 LD₅₀ (Wilcoxon Signed Rank test p<0.0001).</p

Clinical progression of disease after inoculation with the MicroSprayer®.

<p>Clinical progression of disease after inoculation with the MicroSprayer®.</p

Viability of <i>B. pseudomallei</i> (<i>Bp</i>) and <i>B. mallei</i> (<i>Bm</i>) upon use of the MicroSprayer® and delivery of viable organisms into the murine lungs.

<p>Panel A: Bacteria were suspended in PBS to an optical density of 1X10⁶ bacteria/mL, serially diluted, and plated onto agar medium to calculate the number of viable organisms in 50 µL (black bars). The MicroSprayer® was then used to deliver 50 µL of bacterial suspensions into 1 mL of sterile PBS, which was serially diluted and plated onto agar medium to determine the number of viable bacteria (grey bars). Results are expressed as the mean (± standard error) colony forming units (CFU). These experiments were performed in triplicate on 2 separate occasions. Panel B: Bacteria were suspended in PBS to optical densities of 1X10⁶ (Dose 1), 1X10⁵ (Dose 2), and 1X10⁴ (Dose 3) bacteria/mL, serially diluted, and plated onto agar medium to calculate the number of viable organisms in 50 µL (black bars). The MicroSprayer® was then used to deliver 50 µL of bacterial suspensions into the lungs of mice (n=3 per dose). Thirty minutes post-inoculation, the mice were euthanized and their lungs were collected, homogenized, diluted, and plated onto agar medium to determine bacterial loads (grey bars). Results are expressed as the mean (± standard error) CFU. These experiments were performed on at least 2 separate occasions. The Mann-Whitney test was used to compare the number of viable organisms in 50 µL of bacterial suspension (i.e. before using the MicroSprayer®; black bars) to that in 1 mL PBS (panel A) or lung homogenates (panel B) after the use of the MicroSprayer® (i.e. grey bars). No statistically significant differences were noted.</p

LD₅₀ of <i>B. pseudomallei</i> 1026b (<i>Bp</i>) and <i>B. mallei</i> ATCC23344 (<i>Bm</i>) after inoculation with the MicroSprayer®.

<p>The MicroSprayer® was used to deliver the indicated number of bacterial CFU into the lungs of BALB/c mice. Animals were then monitored for clinical signs of illness and morbidity. Survival data were analyzed with the Kaplan-Meier method and the LD₅₀ values were calculated according to Reed and Muench [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076804#B55" target="_blank">55</a>]. The number of animals/group is shown in parentheses. Control mice were inoculated with 50 µL of PBS using the MicroSprayer®. Panels A and B show 2 separate experiments to determine the LD₅₀ of <i>B. pseudomallei</i> strain 1026b. Panels C and D show 2 independent experiments to determine the LD₅₀ of <i>B. mallei</i> ATCC23344. With the exception of the survival curves for PBS and the lowest inoculating CFU dose, survival curves were found to be statistically different using the Logrank test for trend (panels A through D, p ≤ 0.05).</p

ELISA with sera from mice that survived aerosol challenge with lethal doses of <i>B. pseudomallei</i> 1026b (<i>Bp</i>) and <i>B. mallei</i> ATCC23344 (<i>Bm</i>).

<p>Serum samples were serially diluted and placed in duplicate wells of plates coated with CPS (panel A), OPS (panel B), His-tagged BoaA (panel C), His-tagged BPSL1631-BMA1027 (panel D), and His-tagged BPSS0908-BMAA1324 (panel E). Goat α-mouse Abs conjugated to HRP were used as secondary Abs. The y-axis shows absorbance at a wavelength of 650 nm, which is indicative of antibody binding to antigens coating the plates. The x-axis represents serial two-fold dilutions of sera starting at 1:100 to 1:12,800. The results are expressed the mean absorbance (± standard error). Open diamonds show sera from mice that survived challenge with <i>Bm</i>. Closed circles show sera from mice that survived challenge with <i>Bp</i>. Blue squares represent sera from control mice that were inoculated with 50 µL of PBS using the MicroSprayer®.</p

Bacterial loads in bronchoalveolar lavage fluids (BALF) and lungs after inoculation with the MicroSprayer®.

<p>Bacteria were suspended in PBS to optical densities of 1X10⁶ (Dose 1), 1X10⁵ (Dose 2), and 1X10⁴ (Dose 3) bacteria/mL, serially diluted, and plated onto agar medium to calculate the number of viable organisms in 50 µL. The MicroSprayer® was then used to deliver 50 µL of bacterial suspensions into the lungs of mice (n=3 per dose). Control mice were inoculated with 50 µL of sterile PBS. Seventy-two hours post-inoculation, the animals were euthanized and tissues (BALF, lungs) were collected, homogenized, diluted, and plated onto agar medium to determine bacterial loads. Results are expressed as the mean (± standard error) total CFU/per tissue. <i>Bp</i>= <i>B. pseudomallei</i> 1026b, <i>Bm</i>=<i>B. mallei</i> ATCC23344. Black bars= lung homogenates, grey bars=BALF.</p