Delivery to the lower respiratory tract is required for effective immunization with Newcastle disease virus-vectored vaccines intended for humans

Abstract

Newcastle disease virus (NDV), an avian virus, is being evaluated for the development of vectored human vaccines against emerging pathogens. Previous studies of NDV-vectored vaccines in a mouse model suggested their potency after delivery by injection or by the intranasal route. We compared the efficacy of various routes of delivery of NDV-vectored vaccines in a non-human primate model. While delivery of an NDV-vectored vaccine by the combined intranasal/intratracheal route elicited protective immune responses, delivery by the subcutaneous route or the intranasal route alone elicited limited or no protective immune responses, suggesting the necessity for vaccine delivery to the lower respiratory tract. Furthermore, direct comparison of a vaccine based on an NDV mesogenic strain (NDV-BC) with a similarly designed NDV vector based on a modified lentogenic strain carrying a polybasic F cleavage site (NDV-VF) suggested that the two NDV strains were similar in immunogenicity and were equally protective.

Fig. 1

Serum antibody responses following SC or IN/IT immunization of AGM with NDV-BC/HN. (A) NDV-specific and (B) HPIV3 HN-specific HAI serum antibody responses in monkeys following one or two doses of the indicated virus delivered by the SC or IN/IT route. The mean log₂ titer ± SE is shown for each group. Dashed lines represent the limit of detection (2 log₂). Titers below the limit of detection were assigned a value of 1 log₂ for calculation of the mean and SE. Each group contained 4 animals.

Fig. 2

NDV vector shedding following IN or IN/IT immunization of AGM with NDV-BC, NDV-BC/S or NDV-VF/S. TL samples were taken on days 2, 4, and 6 following administration of the indicated NDV construct on day 0. The NDV titer in each sample was determined by plaque titration on monolayers of DF-1 cells. The individual value for each animal is plotted (the NDV-BC control group contained 2 animals; the other groups contained 4 animals each), with the group mean indicated by a horizontal bar. The dotted line represents the limit of detection (0.4 log₁₀ PFU/ml). Values below the limit of detection were assigned a value of 0.2 log₁₀ PFU/ml. This experiment is continued in Fig. 3, Fig. 4, Fig. 5, Fig. 6 and Table 1.

Fig. 3

Serum and mucosal antibody responses following IN or IN/IT immunization of AGM on days 0 and 28 with NDV-BC, NDV-BC/S or NDV-VF/S. (A) NDV-specific HAI serum antibody responses; mean titer ± SE for each group. Values below the limit of detection (2 log₂, dotted line) were assigned a value of 1 log₂ for calculations. (B) SARS-CoV-specific serum IgG responses evaluated by ELISA with purified SARS-CoV S as an antigen; mean fold increase relative to day 0 ± SE for each group. The value for each animal was recorded as the log₂ of the serum dilution resulting in an absorbance at 450 nm that was more than double the background and greater than 0.3. Values below the limit of detection were assigned a value of 2 log₂. At day 0, the mean background log₂ titer for each group was as follows: NDV-BC IN/IT, 2.0; NDV-BC/S IN/IT, 4.3; NDV-VF/S IN/IT, 4.8, NDV-BC/S IN, 2.5. P values were calculated using a repeated measures two-way ANOVA with a Bonferroni post hoc analysis compared to the value for the NDV-BC IN/IT (control) group at the same time point: *P < 0.01. (C) SARS-CoV-neutralizing serum antibody responses; mean log₂ titer ± SE for each group. Values below the detection limit (2 log₂, dotted line) were assigned a value of 1 log₂. P values were calculated using a repeated measures two-way ANOVA with a Bonferroni post hoc analysis compared to the value for the NDV-BC IN/IT (control) group at the same time point: *P < 0.01; **P < 0.001. Note: the titer was not determined for the NDV-BC IN/IT group on days 21 and 49. At these time points, the P values for the other groups were calculated using the day 0 value for the NDV-BC IN/IT group. (D) SARS-CoV-specific mucosal IgA responses; mean increase relative to day 0 ± SE fold for each group. TL samples were collected and concentrated 20–30-fold. They were then analyzed in an IgA isotype-specific ELISA against purified SARS-CoV S starting at a dilution of 1:10 (3.3 log₂). The value for each animal was recorded as the log₂ of the serum dilution resulting in an absorbance at 450 nm that was more than double the background and greater than 0.1. Values below the limit of detection were assigned a value of 2.3 log₂. To account for variability in sample collection and concentration, the titers were then normalized to the total IgA mass in each sample, as determined in a quantitative total IgA ELISA. At day 0, the mean normalized log₂ background titer for each group was as follows: NDV-BC IN/IT, 1.7; NDV-BC/S IN/IT, 1.8; NDV-VF/S IN/IT, 2.0, NDV-BC/S IN, 1.7.

Fig. 4

SARS-CoV challenge virus replication in animals immunized on days 0 and 28 by the IN or the IN/IT routes with NDV-BC, NDV-BC/S or NDV-VF/S: SARS-CoV shedding in respiratory secretions. The animals were challenged with SARS-CoV on day 56, and NW and TL samples were collected on days 1 and 2 post-challenge and analyzed by virus titration in Vero cells. The mean log₁₀ titer ± SE for each group is shown. The limit of detection (1 log₁₀ TCID₅₀/ml) is shown by the dotted line; samples below the limit were assigned a value of 0.5 log₁₀. Mean ± SE values are shown. P values were calculated by comparing the mean titer of the experimental group to that of the control group using a repeated measures two-way ANOVA with a Bonferroni post hoc analysis: *P < 0.05, **P < 0.01.

Fig. 5

SARS-CoV challenge virus replication in animals immunized on days 0 and 28 by the IN or the IN/IT routes with NDV/BC, NDV-BC/S or NDV-VF/S: direct quantitation of SARS-CoV challenge virus in respiratory tract tissue samples (this is a continuation of the experiment shown in Fig. 4). The animals were challenged with SARS-CoV on day 56. They were then euthanized on day 2 post-challenge and tissue samples were taken from the indicated regions. Duplicate tissue samples from each animal were homogenized and the viral titers were determined. The mean log₁₀ titer ± SE for each group is shown. The limit of detection (2 log₁₀) is indicated by a dotted line; titers below the limit were assigned a value of 1.5 log₁₀ TCID₅₀/g. Mean ± SE values are shown. P values were calculated by comparing the mean titer of the experimental group to that of the control group using a repeated measures two-way ANOVA with a Bonferroni post hoc analysis: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6

SARS-CoV challenge of animals immunized on days 0 and 28 by the IN or the IN/IT routes with NDV-BC or NDV-BC/S: IHC and histopathological analysis of the lung tissues harvested 2 days following challenge with SARS-CoV on day 56 (this is a continuation of the experiment shown in Fig. 4, Fig. 5). All micrographs were captured at 400× magnification. Top row: IHC detection of SARS-CoV N protein (S). A significant number of antigen-positive cells are seen in the vector control (left panel), but not the animals immunized with NDV-BC/S by the IN/IT routes (right panel). Antigen-positive cells are also present in animals immunized with NDV-BC/S by the IN route, although at a reduced number (middle panel). Bottom row: Hematoxylin and eosin staining. Features characteristic of SARS-CoV pathogenesis were noted in animals that had been immunized with the empty vector (left panel) or NDV-BC/S by the IN route (middle panel), including marked cellular infiltration (I) of the interstitium and lining epithelium by primarily neutrophils and lymphocytes. In addition, the cilia (C) that are evident in animals previously immunized with NDV-BC/S IN/IT (right panel) are largely absent in animals immunized with the empty vector or with NDV-BC/S IN. Overall, no significant pathological changes were observed in the animals immunized with NDV-BC/S by the IN/IT routes (right panel), whereas animals immunized with NDV-BC/S by the IN route (middle), pathological changes similar to those observed in the empty vector control animal (left) are seen.

Fig. 7

Effect of temperature on plaque formation by NDV-BC. Monolayers of DF-1 (A) and LLC-MK2 (B and C) cells were infected with serial dilutions of NDV-BC (A and B) or HPIV3 (C) and incubated under methylcellulose at the indicated temperatures for 4 (Part A), 10 (part B), and 12 (part C) days. The monolayers were fixed and stained with crystal violet (DF-1 cells), immunostained with anti-NDV antibodies (NDV, LLC-MK2 cells), or immunostained with anti-HPIV3 antibodies (HPIV3). Plaque size was measured using Adobe Photoshop CS2 Extended software. Mean plaque sizes (pixels) ± SE, based on 10 plaques per virus per each temperature are shown.

Fig. 8

Growth kinetics of NDV-BC, NDV-BC/S and HPIV3 in LLC-MK2 cells infected at an MOI of 5 PFU/cell (A) or 0.001 PFU/cell (B) at 32 and 39 °C. Triplicate monolayers of LLC-MK2 cells were infected at the indicated MOI and the virus present in the supernatant was titered at various time points post-infection. The titers were recorded as the log₁₀ PFU/ml ± standard error.

Fig. 9

Expression of NDV antigen in infected LLC-MK2 cells incubated at 32 °C or 39 °C at (A) 24 h after infection with NDV-BC or NDV-BC/S at MOI of 5 PFU, or (B and C) 24, 48, 72 and 96 h after infection with NDV-BC (B) or NDV-BC/S (C) at MOI of 0.001 PFU. Cell lysates were prepared at the indicated time points post-infection and subjected to SDS-PAGE/Western blot analysis using NDV-specific and β-actin specific antibodies. HPI: h post-infection.