Cellular and Humoral Immune Responses to Avian Paramyxoviruses in Chickens

Abstract

The current dissertation (i) examined the effectiveness of a recombinant Newcastle disease virus (NDV) vaccine candidate, (ii) evaluated mucosal and systemic immune responses induced by NDV in chickens with maternally derived antibodies (MDA), and (iii) assessed wild-bird-origin avian paramyxoviruses type 1 (APMV-1) genotype X as potential vaccine candidates for poultry. (i) We previously demonstrated that a prime-boost regime with an infectious bronchitis virus (IBV) Massachusetts (Mass)-type vaccine and recombinant NDV LaSota (rLS) co-expressing IBV Arkansas (Ark)-type trimeric spike ectodomain (Se) and granulocyte macrophage colony stimulating factor (GMCSF) enhances heterologous protection against virulent Ark-type challenge. Here we evaluated protection against Ark-type challenge conferred by administering the rLS/ArkSe.GMCSF and the attenuated Mass viruses simultaneously as a combined vaccine. Protection conferred by the combined vaccine was compared to protection induced by a commercial attenuated ArkDPI (Delmarva Poultry Industry) vaccine as well as by the attenuated Mass vaccine alone. Vaccination with the combined vaccine (rLS/ArkSe.GMCSF + Mass) as well as Mass alone provided significantly less protection against Ark challenge compared to the control using attenuated live ArkDPI vaccine. Only ArkDPI-vaccinated chickens exhibited “sterilizing immunity,” i.e., no virus isolated from ≥10% of chickens after challenge. Chickens vaccinated with the combined vaccine rLS/ArkSe.GMCSF + Mass showed significantly less tracheal damage after challenge than birds vaccinated with the attenuated Mass vaccine alone. In addition, the combined vaccine also resulted in lower rate of virus isolation from the trachea. We concluded that the combined vaccine containing the recombinant virus, and the attenuated Mass enhanced the cross-protective ability of the attenuated Mass vaccine against heterologous challenge. (ii) The immune responses in the Harderian gland (HG) were characterized after NDV LaSota ocular vaccination in antibody naïve specific pathogen free (SPF) chickens and in chickens of commercial origin with NDV MDA. Ocular LaSota vaccination of 13-day-old white-leghorn SPF chickens elicited serum antibody levels that consistently increased after day 15 post-vaccination, while the specific IgA response in lacrimal fluids was already detectable on day 10 after vaccination. Eleven days post-vaccination, the relative abundance of B cells as well as T-helper (CD4+), and cytotoxic T cells (CD8+) in HGs was significantly increased achieving maximum frequencies 16 days post-vaccination. In a second experiment, chickens with MDA originating from NDV-vaccinated commercial white-leghorn layer breeders as well as white-leghorn SPF chickens were vaccinated with NDV LaSota. The LaSota virus successfully replicated in periocular tissues and in the trachea both in commercial and control SPF chickens after vaccination at 2 or 15 days of age (DOA). Vaccination at 2 DOA did not induce a serum NDV antibody response in chickens of commercial origin. In contrast, seroconversion was elicited in commercial chickens upon vaccination at 15 DOA, likely associated with waning of MDA. Unlike systemic IgG responses, vaccination at 2 or 15 DOA elicited strong specific IgA responses in lacrimal fluid in commercial chickens. The IgA response was highest 9 days after vaccination and showed a tendency to decline on day 15 post-vaccination. Commercial chickens vaccinated on day 2 of age showed increased B cells in HG both on days 10 and 16 post-vaccination. The expansion of B cells in the HG in these chickens is consistent with increased IgA levels detected in lacrimal fluids. In contrast, control SPF chickens showed a more limited B cell expansion in HG and lower IgA levels. Vaccination on day 15 of age also triggered a greater increase of B cells in HGs in commercial chickens than in control SPF chickens. The B cell response was accompanied by T helper (CD4+) cell expansion occurring both in commercial and control SPF chickens. These cells expanded to a lesser extent when vaccination was performed at 2 DOA compared to vaccination at 15 DOA. Cytotoxic T cells (CD8+) showed significant expansion irrespective of vaccination day and without differences detected between control SPF chickens and chickens with MDA. We conclude that NDV LaSota elicits vigorous humoral and cell immune responses in the HG. Furthermore, unlike the interference shown by MDA on vaccine-induced serum antibody responses, MDA do not interfere with the mucosal immune response of the HG. (iii) We examined the replication and adaptation of APMV-1 using four isolates: Mallard/US(OH)/04-411/2004, Northern pintail/US(OH)/87-486/1987, Mottled duck/US(TX)/TX01-130/2001, and Mallard/US(MN)/MN00-39/2000, and assessed their potential as vaccine candidates for chickens. The adaptability of each virus was examined by serial passages in embryonated chicken eggs (ECE) and in Vero cells. All APMVs successfully replicated in ECE. In contrast, two isolates passaged in Vero cells showed successful replication and two showed a continuous decline in viral load during passages. Whole genome sequencing analysis identified 14 genomic positions with significant variation in mean allele frequency. Changes of the predominant virus population were characterized by shifts of amino acid (aa) frequency at seven positions. Notably, four of these changes were located in the HN protein, one in matrix (M) protein, and two in the L-protein sequences. Remarkably, while the percentage of alternative amino acids in viral populations passaged in ECE showed limited variation, e.g., at aa position 127 of HN, the frequency varied from 7.4% to 19.8% and HN aa position 192 from 5.1% to 43.5%, the variation of the viral populations passaged in Vero cells was significantly higher at the same positions (e.g. the frequency of the alternative amino acids at HN aa positions 127 and 192 changed from 20.8% to 95.2% and 7.2% to 91.2%, respectively). Isolate 2 passaged in Vero cells displayed a marked variation in alternative amino acid frequencies, specifically at positions 127 within the HN- and 100 within the M- proteins. Isolate 3, while showing no alterations at the same HN positions, showed a considerable change in alternative amino acid frequency in the L protein at position 1875, a change occurring only in the Vero cell environment. One-day-old SPF chickens inoculated with isolates passaged in ECE elicited serum antibody responses similar to those elicited by the LaSota reference strain. In contrast, APMVs passaged in Vero cells showed limited replication in chickens and reduced induction of systemic antibodies. Interestingly, one virus passaged in ECE and another in Vero cells elicited IgA levels in lacrimal fluid comparable to the LaSota strain. We concluded that the four wild-bird APMV isolates tested demonstrated successful adaptation to ECE, with one isolate eliciting overall immune responses comparable to the LaSota virus, supporting their potential as vaccine candidates.