Aerosolized Ebola vaccine protects primates and elicits lung-resident T cell responses (original) (raw)
Study 1
Systemic and mucosal antibody– and cell-mediated responses to aerosol vaccination. Study 1 was designed to compare the EBOV-specific mucosal and systemic antibody and cell-mediated responses in rhesus macaques induced by respiratory tract vaccination with HPIV3/EboGP delivered as an aerosol or as a liquid or i.m. delivery of VRP expressing EBOV GP (ref. 25 and Figure 1A). Groups of rhesus macaques received 2 doses of HPIV3/EboGP or the control empty HPIV3 vector (wild-type virus) by i.n./i.t. delivery at 107.3 PFU per site in the form of a liquid. While aerosol particles less than 3 μM in diameter readily penetrate the small airways (26), the recipients of aerosolized HPIV3/EboGP received a 10-fold higher dose of the vaccine to account for a 10% delivery efficiency of aerosol particles to the lower respiratory tract in primate models due to small lung size and shallow breathing patterns (27, 28) further exacerbated by anesthesia during vaccination (26). The VRP group was vaccinated by 2 sequential s.c. injections of 1010 infectious units of the construct, the optimal regimen protective against EBOV exposure in cynomolgus macaques (ref. 29, G.G. Olinger, unpublished observations); in recent studies, accelerated protection of cynomolgus macaques after a single dose was achieved (30). To analyze the systemic and mucosal antibody responses, we collected the serum separated from peripheral blood and bronchoalveolar lavages (BAL), respectively, from all animals, with the exception of BAL from those in the VRP vaccine group, which were not expected to develop a respiratory mucosal antibody response. Animals were euthanized on day 56, and mononuclear cells were isolated from the lung and spleen tissues and blood to determine response differences from lung-residing T cells versus T cells from the periphery based on route of vaccination and type of vaccine.
Schedules of vaccinations, biosamplings, and EBOV exposure. (A) Study 1: NHP immune response to vaccination. Groups of rhesus macaques were vaccinated with HPIV3/EboGP as an aerosol (n = 4; green) or a liquid via the i.n./i.t. (n = 4; red) route, the empty HPIV3 vector control (n = 2; black), or the VRP vaccine by the i.m. route (n = 4; blue). Twenty-eight days after the first dose, all NHPs received a second dose of their respective vaccine. On day 56, NHPs were euthanized and mononuclear cells were extracted. (B) Study 2: testing of protective efficacy. Groups of rhesus macaques were vaccinated with 1 (n = 4; purple) or 2 doses (n = 4; green) of aerosolized HPIV3/EboGP, 2 doses of liquid HPIV3/EboGP (n = 2; red), or HPIV3 control (n = 2; black). Fifty-five days after vaccination, NHPs were infected with EBOV. At the end of the study, surviving animals were euthanized and terminal bleed samples were collected. Over the course of the 2 studies, serum and BAL samples were collected on indicated days.
Aerosol vaccination induces the robust systemic antibody responses. Analysis of antibody responses by ELISA demonstrated detectable titers of EBOV-specific IgG and IgA in animals vaccinated with HPIV3/EboGP in a liquid or aerosolized form starting at day 14 after vaccination, with a small increase by day 28 (Figure 2, A and B). Administration of the second dose, on day 28, resulted in a strong increase in antibody levels by day 42. Compared with HPIV3/EboGP vaccination, VRP induced lower levels of IgG and IgA on day 14. However, titers reached parity following the second dose.
Serum IgG and IgA response in NHPs from vaccination study 1. NHPs received 2 doses of aerosolized (n = 4; green) or liquid (n = 4; red) HPIV3/EboGP, VRP vaccine (n = 4; blue), or the HPIV3 control (n = 2; black). EBOV-specific serum (A) IgG and (B) IgA were analyzed by ELISA. Values are shown for individual animals in each vaccine group with horizontal bars representing group means. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by 2-way ANOVA with Tukey’s post-hoc test. For clarity, comparisons to VRP on days 14 and 28 are shown.
Surface plasmon resonance (SPR) analysis of total EBOV-binding antibody (Supplemental Figure 1, A and B; supplemental material available online with this article; doi:10.1172/JCI81532DS1) revealed a robust response in HPIV3/EboGP-vaccinated animals after dose 1 (day 28), which slightly increased after dose 2 (day 56) to yield somewhat higher levels in aerosol recipients. Compared with HPIV3/EboGP recipients, VRP-vaccinated animals exhibited weaker EBOV antibody–binding profiles; 3-fold fewer EBOV-binding antibodies were generated after dose 1, but numbers rose after dose 2 so that they were marginally lower than levels in HPIV3/EboGP recipients. Antibody avidity was determined by analysis of antibody dissociation rates (off-rate), where a low value was indicative of higher avidity (Supplemental Figure 1C). After dose 1, the dissociation rates of antibodies from aerosol and liquid HPIV3/EboGP-vaccinated animals were equal and lower than those of VRP-vaccinated animals, suggesting that higher avidity antibodies were generated by the respiratory vaccine. The VRP group displayed a more heterogeneous antibody off-rate profile. The second vaccine dose did not alter the dissociation rates of antibodies from HPIV3/EboGP-vaccinated animals. In contrast, the dissociation rate of antibodies from each VRP-vaccinated animal was reduced, with 3 out of 4 animals exhibiting rates equal to those observed in HPIV3/EboGP-vaccinated animals.
Testing of the ability of sera to neutralize EBOV in vitro demonstrated comparable neutralizing titers in animals vaccinated with either forms of HPIV3/EboGP, which reached high levels after dose 1 (mean titers 1:460 and 1:250 for aerosolized and liquid forms, respectively) and were only slightly increased after dose 2 (Figure 3A). Following vaccination with VRP, neutralizing titers were low after dose 1, but increased to levels comparable to those in HPIV3/EboGP-vaccinated animals after dose 2. Taken together, these data suggest that vaccination with aerosolized HPIV3/EboGP induces systemic antibody responses comparable to those after respiratory vaccination with liquid HPIV3/EboGP. The data also show that following dose 1, the antibody response to HPIV3/EboGP was greater in magnitude and avidity than that to VRP.
Serum-neutralizing antibody responses in NHPs from vaccination study 1. NHPs received 2 doses of aerosolized (n = 4; green) or liquid (n = 4; red) HPIV3/EboGP, VRP vaccine (n = 4; blue), or the HPIV3 control (n = 2; black). Serum-neutralizing antibody responses against (A) EBOV, (B) BDBV, and (C) SUDV were determined by plaque-reduction assays. Values are shown for individual animals in each vaccine group, with horizontal bars representing group means. **P < 0.01; ***P < 0.001; ****P < 0.0001, by 2-way ANOVA with Tukey’s post-hoc test.
Two vaccine doses induce neutralizing antibodies against heterologous EBOVs. The breadth of the antibody response was determined by the ability to cross-neutralize EBOVs Bundibugyo (BDBV) and Sudan (SUDV). Following dose 1, only 1 HPIV3/EboGP liquid and 1 VRP recipient demonstrated detectable BDBV-neutralizing titers of 1:33 and 1:15, respectively. However, after dose 2, comparable titers were achieved in most animals from all vaccine groups (Figure 3B). In contrast to BDBV, SUDV-neutralizing antibodies were detected in most animals after dose 1 and in all animals except 2 liquid HPIV3/EboGP recipients after dose 2 (Figure 3C). Compared with the HPIV3/EboGP vaccine, 2 doses of VRP yielded markedly higher BDBV- and SUDV-neutralizing titers. Taken together, these data suggest that antibody cross-neutralization is stronger following administration of 2 vaccine doses and that the breadth of the response has considerable animal-to-animal variability.
Respiratory vaccination induces mucosal antibodies in the respiratory tract. To analyze mucosal antibody responses in the respiratory tract, concentrated BAL collected from animals vaccinated with either forms of HPIV3/EboGP or the empty HPIV3 vector were analyzed by ELISA and virus neutralization assays. High titers of IgG and IgA were detected in aerosol and liquid HPIV3/EboGP recipients after dose 1, and titers further increased following dose 2 (Figure 4, A and B). The mean neutralizing antibody titers in aerosolized and liquid recipients reached 1:9 and 1:23, respectively, after dose 1 and 1:112 and 1:47 after dose 2 (Figure 4C). Thus, aerosol vaccination induces robust mucosal antibody responses in the respiratory tract.
Mucosal antibody responses to HPIV3/EboGP in NHPs from vaccination study 1. NHPs received 2 doses of aerosolized (n = 4; green) or liquid (n = 4; red) HPIV3/EboGP or the HPIV3 control (n = 2; black). EBOV-specific (A) IgG and (B) IgA were determined by ELISA, and (C) EBOV-neutralizing antibodies were determined by a plaque-reduction assay. Values are shown for individual animals in each vaccine group, with horizontal bars representing group means. For clarity, comparisons on day 21 made with 2-way ANOVA with Tukey’s post-hoc test are shown. **P < 0.01; ***P < 0.001; ****P < 0.0001.
Respiratory vaccination induces a more robust T cell response in the lungs than in blood and spleen. Mononuclear cells derived from tissues of the lungs, blood, and spleen were stimulated with peptides spanning the entire EBOV GP, and CD8+ T cell subpopulations positive or negative for CD103, a marker highly expressed by mucosal CD8+ T lymphocytes, and CD4+ T cells, were analyzed for functional markers of activation by flow cytometry (Figure 5). The magnitude of both the CD8+ and CD4+ T cell response induced by HPIV3/EboGP was dramatically greater in the lungs than in the blood or spleen. For example, in aerosolized HPIV3/EboGP recipients, the mean percentages of lung CD103+CD8+ T cells secreting TNF-α were 15- and 28-fold greater than in the blood and spleen, respectively. The mean percentages of lung CD4+ T cells secreting TNF-α were 12- and 14-fold greater than in blood and spleen, respectively. These data suggest the cell-mediated response occurs predominately in the respiratory tract, the site of HPIV3/EboGP replication, with limited, but detectable, systemic spread of these virus-specific lymphocytes.
Cell-mediated response in vaccine recipients from study 1. NHPs were vaccinated with HPIV3/EboGP aerosol (n = 4; green symbols), HPIV3/EboGP liquid (n = 4; red), the VRP vaccine (n = 4; blue), or HPIV3 control (n = 2; black). Magnitude of CD8+ CD103+ (left) and CD103– (middle) T cell subsets and CD4+ T cells (right) from the (A and D) lungs, (B) blood, and (C) spleen positive for markers of activation following GP peptide stimulation, displayed as (A–C) a percentage of total CD8+ or CD4+ cells or (D) level of expression (MFI). The group means are shown by horizontal bars. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by 2-way ANOVA with Tukey’s post-hoc test.
The activated CD8+ cells are almost equally divided between the CD103+ and CD103– populations. CD103 is expressed at high levels by mucosal CD8+ T cells, specifically those associated with lung tissue, and is involved with their mucosal homing, cytotoxicity against respiratory epithelial cells, and retention as effector memory T cells (31). We therefore compared distribution of the activated CD103+CD8+ T cells and their CD103– counterparts at 3 different sites. In lungs, the activated CD8+ cells were almost equally distributed as CD103+ and CD103– populations (Figure 6). Contrary to our expectations, the frequency of activated CD103– populations in the blood and spleen was only marginally higher than that of the CD103+CD8+ T cells (Figure 6). These data suggest that the CD103+ fraction of activated CD8+ T cells may be active not only at mucosal sites but also in the periphery.
Polyfunctional CD8+ T cell response from vaccination study 1. NHPs were vaccinated with aerosol (n = 4; green) or liquid (n = 4; red) HPIV3/EboGP, VRP (n = 4; blue), or HPIV3 control (n = 2; black). Upper panels: percentage of CD103+ and CD103– CD8+ T cell subsets in the lungs, blood, and spleen of each animal, producing all possible combinations of activation markers. Lower panels: proportion of response patterns for each animal, grouped according to the number of positive markers whereby pie slices represent all 4 (4+, dark red) or any combination of 3 (3+), 2 (2+), or 1 (1+) of the measured markers (the lighter colors). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by 2-way ANOVA with Tukey’s post-hoc test.
Respiratory vaccination induces a greater activation of CD8+ T cells in lungs than in blood and spleen. The majority of EBOV-specific lung-resident CD8+ T cells induced by HPIV3/EboGP were polyfunctional, i.e., positive for 2 or more of the 4 markers of activation (CD107a, IFN-γ, IL-2, and TNF-α). Cells expressing all 4 markers of activation were rare, observed mainly in recipients of the liquid form of HPIV3/EboGP (Figure 6). IFN-γ+IL-2+TNF-α+ in the absence of CD107a dominated the triple-marker functional responses. The double-positive cell populations were generally characterized by cells producing IFN-γ in combination with TNF-α or IL-2. Small numbers of cells possessed an IL-2+TNF-α+ phenotype, while combinations with CD107a were only elicited by 2 animals, which received the liquid HPIV3/EboGP vaccine. There was no clear association between the polyfunctional response pattern in the lungs versus the spleen and blood. Moreover, the proportion of the polyfunctional response was generally lower in the blood and spleen, with the majority of cells positive for only a single marker of activation and varying trends among the respiratory vaccinees (Figure 6). These data suggest that respiratory vaccination with HPIV3/EboGP not only induces a greater number of CD8+ T cells in lungs than in blood and spleen, but also results in a greater activation of lung CD8+ T cells.
Respiratory vaccination induces a greater activation of CD4+ T cells in lungs and spleen than in blood. HPIV3/EboGP-activated CD4+ T cells were analyzed for the production of any combination of IFN-γ, IL-4, IL-17, and TNF-α. At least 25% of cells in lungs were positive for 2 markers of activation, namely TNF-α and IFN-γ (Figure 7). Other 2-functional response combinations were not detected. A strong monofunctional response pattern was also observed, dominated by the expression of TNF-α, a smaller fraction of IFN-γ+ cells, and the absence of IL-4+ cells. An analogous phenotypic pattern of activation markers was observed in the blood. However, bifunctional TNF-α+IFN-γ+ cells were detected at a much lower frequency, suggesting either a lower activation of CD4+ T cells circulating in the blood or the retention of resident CD4+ T cells in the lung serving as protectors against respiratory infections (Figure 7). Interestingly, the number of activated CD4+ T cell populations was greater in the spleen than in the blood and mostly composed of either bifunctional TNF-α+IFN-γ+ populations or monofunctional IFN-γ, TNF-α, or IL-17 CD4+ T cells. However, the frequency of these functional populations varied greatly among individual animals from each group (Figure 7). Following peptide stimulation, the level of IL-4 expression was significantly less in CD4+ T cells isolated from the spleens of aerosol vaccinees compared with the control and VRP recipients (Figure 5C and Figure 7). This may be an indication of HPIV3/EboGP ability to produce a predominantly Th1 response against infection, while IL-4 expression in control animals may represent a background or inadequate response to peptide stimulation. Taken together, these data suggest that respiratory vaccination with HPIV3/EboGP induces the most potent CD4+ T cell response in lungs, followed by spleen, and a substantially lower response in the peripheral blood.
Multiple effector functions of CD4+ T cells in study 1 vaccine recipients. CD4+ cell response profiles in lungs, blood, and spleen of aerosol (n = 4; green) or liquid (n = 4; red) HPIV3/EboGP, VRP vaccine (n = 4; blue), or HPIV3 control (n = 2; black) recipients from study 1 are shown. The percentage of CD4+ T cells eliciting all possible functional response combinations of IFN-γ, IL-4, IL-17, and TNF-α (dot plots) and their proportional contribution (pie charts) to the total EBOV GP–induced response are displayed in the same manner as in Figure 6. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by 2-way ANOVA with Tukey’s post-hoc test.
Vaccination induces Th17 cells mostly in spleen. The role of Th17 in protection against viral infections is not clear: while some studies demonstrated their role in protection (32, 33), others suggested no role (34) or even enhancement of viral infection (35). Development of Th17 cells was previously associated with pulmonary immune defense (36) or inflammation in chronic obstructive pulmonary disease and lung cancer (37). Modest amounts of GP-specific CD4+ T cells positive for IL-17 were detected in spleens of some of the vaccinated animals irrespective of the vaccine type and were rare in the lungs and blood (Figures 5 and 7), with no IL-17–inclusive activation marker combinations dominant in any of the 3 sites. The majority of IL-17–positive cells from the spleen were monofunctional (Figure 7). Only a few animals, specifically from the VRP group, yielded very low percentages of cells positive for IL-17 in combination with IFN-γ and IL-4 or IFN-γ only. While IL-17+IFN-γ+ cells correspond to the previously established Th17/Th1 phenotype (38), we are unaware of any previous identification of Th17 cells secreting IL-4. The protective role of Th17 cells induced by EBOV vaccines requires additional studies.
Delivery of HPIV3/EboGP in aerosolized form increases the T cell response in lungs. We next compared lung CD8+ and CD4+ T cell responses to liquid and aerosol administration of HPIV3/EboGP. Strikingly, IFN-γ+IL-2+TNF-α+ and IFN-γ+TNF-α+ CD8+ T cells were above 0.1% in all 4 aerosolized HPIV3/EboGP recipients, but only in 2 out of 4 i.n./i.t.–vaccinated animals (Figure 6). More aerosol recipients yielded higher percentages of lung CD8+ T cells bifunctional for IFN-γ+IL-2+ and CD103+CD8+ T cells monofunctional for IFN-γ or TNF-α than seen in liquid vacinees. Interestingly, functional response combinations incorporating CD107a were mostly evident in some of the liquid HPIV3/EboGP vaccine recipients: their lung CD103+ or CD103–CD8+ T cells were capable of CD107a mobilization. This trend was observed in the blood and spleen of these liquid recipients, though fewer CD103+ cells were mobilizing CD107a (Figures 5 and 6). Lung CD103–CD8+ T cells in aerosolized HPIV3/EboGP recipients expressed more TNF-α than those of the liquid recipients (Figure 5D) even though the total cell numbers were comparable between these groups (Figure 5A).
While the total amount of CD4+ T cells producing IFN-γ or TNF-α and the levels at which they express these cytokines were generally comparable between aerosol and liquid HPIV3/EboGP recipients (Figure 5, A and D), the aerosol group had double the total number of lung CD4+ T cells expressing TNF-α (Figure 5A). Analysis of multifunctional CD4+ T cells demonstrated that bifunctional TNF-α+IFN-γ+ and monofunctional TNF-α+ cells in lungs were 2.1- and 2.9-fold, respectively, greater in the aerosol HPIV3/EboGP group than in the liquid vaccine group (Figure 7). The greater activation of T cells in lungs by aerosolized HPIV3/EboGP may be the consequence of more efficient delivery to the pulmonary bronchiole and more efficient activation of pulmonary antigen-presenting cells, such as CD103+ dendritic cells.
Vaccination with VRP induces T cells responses predominantly in spleen. Comparison of HPIV3/EboGP and VRP demonstrated that in both lungs and blood, greater numbers and proportions of polyfunctional CD8+ and CD4+ T cell populations were induced in most of the HPIV3/EboGP vaccine recipients, while responses from VRP recipients were generally monofunctional and observed in only a few recipients (Figures 6 and 7). The frequency of the response in the VRP-vaccinated animals that did yield multifunctional T cells was somewhat comparable to that in the HPIV3/EboGP recipients (Figures 6 and 7). The functional quality of CD8+ and CD4+ cells from VRP recipients was greater in the spleen, though no discernible pattern in functional marker combinations was observed among the animals (Figure 6). All VRP-vaccinated animals, however, possessed CD103+CD8+ T cells monofunctional for TNF-α. Blood- and spleen-isolated CD8+ T cells from VRP vaccine recipients exhibited greater response than those isolated from the lungs, with the majority being monofunctional.
Polyfunctional T cells secrete greater amounts of IFN-γ in lungs. We next compared the mean fluorescence intensity (MFI) of IFN-γ for CD8+ and CD4+ T cell populations expressing all IFN-γ–inclusive activation marker combinations. We found that IFN-γ expression by CD8+ T cells gradually increased as the number of functional markers increased, with higher MFI detected in populations eliciting all 4 markers of activation, followed by cells positive for 3 markers (IFN-γ+TNF-α+IL-2+), 2 markers (IFN-γ+IL-2+, IFN-γ+TNF-α+), and IFN-γ only (Figure 8A). Interestingly, CD8+ T cells positive for all 4 markers of activation from the aerosol and liquid HPIV3/EboGP groups produced comparable IFN-γ MFI (Figure 8A) despite the scarcity of these cells in the aerosol group (Figure 6). CD8+ T cells from the lungs, blood, and spleen yielded comparable IFN-γ MFI (Figure 8A) despite higher percentages of IFN-γ–positive cells found in the lungs (Figure 5). Similar to the IFN-γ expression trend observed in CD8+ T cells, greater IFN-γ MFI were detected in triple-marker CD4+ T cell populations positive for IFN-γ, TNF-α, and either IL-4 or IL-17 (Figure 8B) despite their rarity (Figure 7). A high IFN-γ MFI was observed in CD4+ T cells coexpressing IFN-γ and TNF-α from the lungs, blood and spleen, while monofunctional cells yielded high IFN-γ MFI only in the lungs of HPV3/EboGP recipients. Taken together, these data suggest that a greater level of activation of cells is accompanied not only by simultaneous secretion of multiple cytokines, but also by the greater level of secretion of IFN-γ by polyfunctional T cells. These data also suggest that respiratory delivery of HPIV3/EboGP induces highly activated T cells in lungs.
IFN-γ expression in polyfunctional T cells. IFN-γ MFI (y axes) for functional response combinations in (A) CD8+ and (B) CD4+ T cells in the lungs, blood, and spleen of aerosol (n = 4; green) or liquid (n = 4; red) HPIV3/EboGP, the VRP vaccine (n = 4; blue), or HPIV3 control (n = 2; black) recipients from study 1. The x axes show combinations of functional markers positive for IFN-γ with CD107a, IL-2, or TNF-α (CD8+) or IL-4, IL-17, or TNF-α (CD4+). IFN-γ MFI for individual animals is shown with horizontal bars representing group means.
Study 2
Protective efficacy of aerosol vaccination. In the second study (Figure 1B), we evaluated protection conferred by aerosolized HPIV3/EboGP administered to rhesus macaques. Due to the high EBOV-neutralizing antibody response induced after the first aerosolized dose (Figure 3A), we included a single-dose group in addition to a 2-dose group. The control groups included animals vaccinated with 2 doses of HPIV3/EboGP delivered to the respiratory tract as a liquid, which were expected to be protected (21), and animals vaccinated with an empty vector. Following vaccinations, BAL and serum were periodically collected for analysis of mucosal and systemic antibody responses, respectively. On day 55 (27 days after the second or single vaccination with aerosolized HPIV3/EboGP), the animals were exposed to 1,000 PFU of EBOV delivered by the i.m. route. Following exposure, blood samples were collected every 2 days after infection to analyze viremia and blood chemistry.
Antibody responses to aerosol vaccination. Prior to infection, the 2-dosing schedule of HPIV3/EboGP (aerosol or liquid) resulted in the induction of serum and EBOV-specific mucosal IgG, IgA, and neutralizing titers (Figure 9) in accordance with the humoral response observed in vaccinated rhesus macaques from study 1 (Figure 2). EBOV-specific serum IgG were detected on day 14 after HPIV3/EboGP vaccination (Figure 9A), and levels increased by day 28. After the second dose, antibody levels further increased and plateaued by day 51, the last time point analyzed. IgG levels were similar in animals vaccinated with liquid and aerosolized HPIV3/EboGP. EBOV-specific serum IgA (Figure 9B) was induced with a kinetic pattern similar to that of IgG. The kinetics of IgG and IgA induction were similar between animals administered only a single aerosol HPIV3/EboGP dose and those that received the initial dose of the 2-dose regime. However, at the time of infection, the levels of IgG and IgA in the single-dose group were somewhat lower than in the 2-dose groups. As in study 1 (Figure 3A), high levels of serum EBOV-neutralizing antibodies were detected 28 days after the first dose of HPIV3/EboGP, delivered in either form, which did not increase further following a second dose (Figure 9C). Equal levels of EBOV-neutralizing antibodies were therefore achieved in all groups, including the single-dose aerosol group, at the time of infection.
Serum and mucosal antibody responses in EBOV infection study 2. One dose of aerosolized HPIV3/EboGP (n = 4; purple), 2 doses of aerosolized HPIV3/EboGP (n = 4; green), 2 doses of liquid HPIV3/EboGP (n = 2; red), and 2 doses of HPIV3 control (n = 2; black) were given. EBOV-specific IgG and IgA were detected in (A and B) serum and (D and E) concentrated BAL fluids by ELISA. EBOV-specific neutralizing antibodies were detected in (C) serum and (F) mucosa by plaque-reduction assays. TB, terminal bleed. Antibody titers are shown for individual animals in the vaccine groups, with the horizontal bar representing the group means. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by 2-way ANOVA with Tukey’s post-hoc test. For clarity, comparisons made are shown only for the final time point.
EBOV-specific mucosal IgG (Figure 9D) and IgA (Figure 9E) were induced after dose 1 (day 21) and marginally increased after dose 2 (day 44), resulting in higher levels in the 2-dose groups compared with the 1-dose aerosol group. In 2-dose groups, mucosal EBOV-neutralizing antibodies were detected after the first dose and were not further augmented by a second dose (Figure 9F). However, in the single-dose aerosol group, mucosal-neutralizing antibodies were detected in only 1 out of the 4 animals despite a robust serum-neutralizing response. This may be explained by inefficient BAL collection or titers below the limit of detection due to the margin between vaccination and BAL collection being 5 fewer days than for animals that received their first inoculation of a 2-dose vaccine regime.
A single dose of aerosolized EBOV/EboGP protects rhesus macaques against EBOV infection. After EBOV infection, the 2 control HPIV3 vector–vaccinated animals demonstrated increased clinical sickness scores. They also demonstrated sharp increases in serum alanine aminotransferase and bilirubin, markers of liver disease, creatinine, marker of kidney disease, and blood urea nitrogen, marker of liver and kidney disease (Figure 10A); all 4 are typically increased during EBOV infections (reviewed in ref. 39). On days 8 and 9 after infection, the animals became moribund and were euthanized (Figure 10B). In contrast, these markers were normal in all HPIV3/EboGP recipients, with the exception of a small transient increase of alanine aminotransferase in 1 recipient of the single aerosol dose on day 10 (Figure 10A). The clinical scores and body temperatures were also normal, with the exception of a small transient increase in an animal vaccinated with 2 doses of aerosolized HPIV3/EboGP, 3 animals vaccinated with 1 dose of aerosolized HPIV3/EboGP, and 1 animal vaccinated with 2 doses of liquid HPIV3/EboGP (Figure 10, C and D). Plaque titration analysis of serum collected every 2 days after EBOV exposure demonstrated high levels of viremia in the 2 control animals (Figure 10E). In contrast, no viremia was detected in any of the vaccinated animals. Analysis of serum by quantitative real-time reverse-transcription–PCR (RT-PCR), however, demonstrated transient low levels of EBOV RNA in some of the vaccinated animals, which completely disappeared by day 10.
EBOV infection of vaccinated NHPs in study 2. HPIV3 control (n = 2; black), 2 doses of HPIV3/EboGP aerosol (n = 4; green), 1 dose of HPIV3/EboGP aerosol (n = 4; purple), or 2 doses of HPIV3/EboGP liquid (n = 2; red) were given. (A) Peripheral blood markers of EBOV disease. The values alanine aminotransferase, total bilirubin, creatinine, and blood urea nitrogen are shown for each animal. X’s indicates euthanasia of the moribund control animals after the last indicated blood sample was collected. (B) Percentage survival over 28 days following i.m. injection of 1,000 PFU of EBOV. (C) Clinical sickness scores ranging from 0–9, where 0–3 require no medical intervention and 9 requires euthanasia. (D) Temperature. (E) Viremia assessed by plaque assay (PFU/ml; symbols) and the levels of viral RNA in serum determined by quantitative RT-PCR (relative PFU/ml; bars). (A and C–E) Values are shown for each animal on days 0, 2, 4, 6, 8,10, 14, 21, and 28 after infection, with the exception of PFU/ml on day 28 (not determined) and relative PFU/ml on days 14, 21, and 28 (not determined).
All 10 surviving HPIV3/EboGP-vaccinated animals were euthanized 28 days after infection. H&E and immunohistochemical staining of the spleen, liver, kidney, and lungs of all HPIV3/EboGP-vaccinated, EBOV-infected macaques confirmed normal tissue histology and the absence of EBOV antigen (spleen and liver from a representative HPIV3/EboGP recipient shown in Supplemental Figure 2). Tissues from the control HPIV3 vector–vaccinated macaques displayed EBOV antigen and histologic lesions consistent with EBOV infection, including necrotizing hepatitis and splenic lymphoid depletion (Supplemental Figure 2). Taken together, these data suggest that a single administration of aerosolized HPIV3/EboGP completely protects animals against death and severe disease caused by a uniformly lethal dose of EBOV administered by the i.m. route.