A centralised immunogen approach to develop a more broadly protective modified live porcine reproductive and respiratory syndrome virus 1 vaccine candidate

Abstract

More efficacious vaccines are required to improve control of porcine reproductive and respiratory syndrome viruses (PRRSV). One strategy that has shown promise is the use of centralized antigens, generated from consensus sequence data. Here, we evaluated the consensus sequence approach to develop a PRRSV-1 modified live virus (MLV) vaccine candidate, 'EU-PRRSV-Con'. EU-PRRSV-Con strain was engineered by inserting consensus sequence open-reading frames encoding envelope proteins of 67 PRRSV-1 strains into an attenuated PRRSV-1 strain backbone. EU-PRRSV-Con was evaluated in pigs and benchmarked against a licensed MLV vaccine. Efficacy was assessed against three different PRRSV-1 isolates. Neutralizing antibodies were elicited by EU-PRRSV-Con, which were more reactive than those induced by the licensed MLV. EU-PRRSV-Con provided better levels of protection (reduced viral loads and lung pathology) than the licensed MLV, although the efficacy against a divergent PRRSV-1 subtype 3 strain was more limited. These data support the development of EU-PRRSV-Con as a vaccine that may aid control of PRRSV-1.

Fig. 1. Assessment of EU-PRRSV-Con replication in MARC-145 cells and porcine BALC.

Replication kinetics of EU-PRRSV-Con, wild type and recombinant PRRSV-1 Olot/91 in MARC-145 cells (A) and BALC (B) cultures following infection with a multiplicity of infection of 0.1. Infectious titres in culture supernatants were determined by subsequent titration on MARC-145 cells. Datapoints represent the mean of technical triplicates and error bars represent the standard deviation (SD). Significant differences (p < 0.05) are described by letters where a: significant difference from wild type PRRSV-1 Olot/91, and c: significant difference from EU-PRRSV-Con. The dashed horizontal line indicates the assay limit of detection.

Fig. 2. Weight gain, clinical signs, and rectal temperatures following immunisation and challenge in all three studies.

On day 0, pigs were vaccinated with either EU-PRRSV-Con or benchmark MLV PRRS or were unvaccinated. On day 35 post-vaccination, pigs were challenged with the PRRSV-1 strains 215-06 (Study 1), 21301-19 (Study 2) or SU1-Bel (Study 3). In studies 2 and 3, groups of vaccinated pigs were left unchallenged. Following immunisation and challenge, animals were weighed on a weekly basis (A–C) and clinical signs (D–F) and rectal temperatures (G–I) were measured daily for fourteen days. Data presented as mean body weight and rectal temperatures (error bars represent the SD) or datapoints representing the clinical scores for individual pigs. Significances (p < 0.05) are described by letters where a: significant difference from EU-PRRSV-Con/challenged; b: significant difference from benchmark MLV/challenged; c: significant difference from unvaccinated/challenged; d: significant difference from EU-PRRSV-Con/no challenge; e: significant difference from benchmark MLV/no challenge.

Fig. 3. Gross lung pathology, histopathology, and viral immunohistochemistry of lungs post-challenge.

Scoring of macroscopic and microscopic lung lesions at 7 DPC was performed blind in study 1 (A), 2 (B) and 3 (C). Each lobe was given a score for lesion coverage, from which each lobe score was weighted based on its relative size and then summed to give the gross lung pathology score. Microscopic lesions were scored using the “Morgan” scoring system on H&E-stained sections and the “Iowa” scoring system, which incorporates the presence of viral antigen, assessed by IHC. Significances (p < 0.05) are described by letters where a: significant difference from EU-PRRSV-Con/challenged; b: significant difference from benchmark MLV/challenged; and c: significant difference from unvaccinated/challenged group.

Fig. 4. RT-qPCR results of serum and nasal swab samples of each group across all studies.

Blood samples and nasal swabs were collected weekly post-vaccination and twice weekly post-challenge. Viraemia (A–C) and virus shedding (D–F) were inferred by RT-qPCR. Results are expressed as the mean data for each group at each timepoint and error bars represent the SD. Significances (p < 0.05) are described by letters where a: significant difference from EU-PRRSV-Con/challenged; b: significant difference from benchmark MLV/challenged; c: significant difference from unvaccinated/challenged; d: significant difference from EU-PRRSV-Con/no challenge; e: significant difference from benchmark MLV/no challenge.

Fig. 5. Assessment of PRRSV-neutralising antibody responses post-vaccination and -challenge.

Serum samples from day 38 and 56 (3 and 21 DPC) were assessed for their ability to neutralise MARC-145 cell infection by vaccine viruses and BALC infection by challenge viruses. Neutralisation of EU-PRRSV-Con by study 1 (A), 2 (B) and 3 (C) sera; benchmark MLV by study 1 (D), 2 (E) and 3 (F) sera; 215-06 by study 1 sera (G); 21301-19 by study 2 sera (H); and SU1-Bel by study 3 sera (I) are shown with datapoints representing individual pig sera, and bars represent median values for each treatment group. Significances (p < 0.05) are described by letters where a: significant difference from EU-PRRSV-Con/challenged; b: significant difference from benchmark MLV/challenged; c: significant difference from unvaccinated/challenged; d: significant difference from EU-PRRSV-Con/no challenge; e: significant difference from benchmark MLV/no challenge.

Fig. 6. Assessment of PRRSV-specific IFN-γ responses post-vaccination and/or -challenge in PBMC, BALC and TBLN cells.

Responses were assessed following PRRSV-1 stimulation of PBMC post-vaccination and/or -challenge in study 1 (A), 2 (B) and 3 (C), post-challenge (7 and 21/22 DPC) of BALC in study 1 (D), 2 (E) and 3 (F), and post-challenge (7 and 21/22 DPC) of TBLN cells in study 1 (G), 2 (H) and 3 (I). Datapoints represent mock-corrected data for individual pigs, line and bars represent the mean values for each treatment group, and error bars show the SD. Significances (p < 0.05) are described by letters where a: significant difference from EU-PRRSV-Con/challenged; b: significant difference from benchmark MLV/challenged; c: significant difference from unvaccinated/challenged; d: significant difference from EU-PRRSV-Con/no challenge; e: significant difference from benchmark MLV/no challenge.