The humoral immune responses to IBV proteins. E. Dan Heller and Rosa Meir The Hebrew University of Jerusalem, Israel COST FA1207 meeting WG2 + WG3, Budapest, Jan. 2015 1
IBV encodes four major structural proteins: the spike protein (S), the phosphorylated nucleo capsid protein (N), the integral membrane protein (M) and the small envelope protein (E). The S protein is initially translated into a precursor glycoprotein that is cleaved post translationally to form two subunits, S1 and S2. 2
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Vaccination with the purified rs1, rn or rm proteins expressed in Escherichia coli. 12 chickens per group vaccinated by eye drop with the purified proteins at: day of age 2 weeks and 5 weeks. At the age of 44 days all the chickens were challenged with 10 5 EID 50 pathogenic M41. Positive control Vaccination: by eye drop with live H120 vaccine was performed twice: at day of age and at 3 weeks of age. 4
Group No. Vaccine Protein concentration Delivery mode 1. 12 chicks PBS Eye drop 2. 12 chicks H120 1 dose / chicken 2 times 3. rs1 150 g / chicken 12 chickens 3 times 4. rn 150 g / chicken 12 chickens 3 times 5. rm 150 g / chicken 12 chickens 3 times Eye drop Eye drop Eye drop Eye drop 5
Protection. vaccination Average Percent protection Non vaccinated 9% 11% 2 X H120 89% 96% 3 X S1 40% 43% 3 X N 30% 33% 3 X M 10% 6
Systemic humoral response. Antibody titers were measured with a commercial kit (IDEXX) Non of the experimental groups demonstrated positive titers before the challenge (Cutoff 397). H120 rs1 rn rm Non vaccinated 640 390 320 211 160 7
9 days post challenge, High antibody titers were observed H120 rs1 rn rm nonvaccinat ed 1720 1260 1450 970 940 8
On the day of challenge HI titers were: H120 rs1 rn rm Non vaccinated 1:360 1:600 1:48 1:1 0 HI titers 9 days post challenge: H120 rs1 rn rm Non vaccinated 1:1024 1:1200 1:240 1:228 1:256 9
IgA and IgG titers were measured by specific ELISA in tracheal washes. Results are given in O.D. 620nm values. IgA H120 S1 N M Non Vaccinated At 14 days of age At 40 days of age At 49 days of age At 58 days of age 0.01 0.08 0.08 0.18 0.13 0.22 0.20 0.16 0.15 0.12 0.54 0.26 0.30 0.16 0.13 0.80 0.70 0.58 0.28 0.24 10
IgG H120 S1 N M Nonvaccinated At 14 days of age At 35 days of age At 49 days of age At 58 days of age 0.02 0.10 0.06 0.05 0.03 0.14 0.18 0.08 0.05 0.03 0.16 0.20 0.16 0.05 0.03 0.38 0.28 0.24 0.15 0.32 11
Summing up the immune response of chickens to rs1 glycoprotein expressed in Escherichia coli as a vaccine applied by eye drops. Challenge 3 weeks after the third vaccination: low protection (40%) as compared to 90% of 2 vaccinations with H120. IBV antibody titers measured by commercial ELISA kit (IDEXX) before challenge were below detectable level. But Nine days post challenge H120= 1720 rs1= 1260 12
Three weeks after the third vaccination a high HI antibody titer was found (1:600) while the titer of the group vaccinated twice with H120 (1:360). rs1 vaccination resulted in virus specific IgA (OD 620 = 0.25) and IgG titers (OD 620 = 0.20) in tracheal washes. After challenge these titers were increased. both lower than the titers resulted from H120 vaccination 13
Summing up the immune response of chickens to rn protein expressed in Escherichia coli as a vaccine applied by eye drops. Challenge 3 weeks after the third vaccination resulted in low protection of 30% IBV antibody titers measured by commercial ELISA kit (IDEXX) of all the groups before challenge were below detection level. Nine days post challenge H120= 1720 rn= 1450 14
rn Three times rn protein vaccination resulted in IgA level of OD 620 = 0.30 and low IgG titers (OD 620 = 0.16) in tracheal washes. The immune responses to the purified M protein were low. Protein M play a negligible role in the humoral immune response to IBV 15
Update literature review S1 16
Already in 1994 Ignjatovic and Galli (Arch.Virol. 138:117) produced the S1, N, and Mproteins from IBV N1/62 which was nephropathogenic and used them for immunization of chickens. Multiple immunization were necessary for induction of an antibody response. Neither the Nnor the Mantigen induced protection to a virulent challenge after 4 immunizations. 17
Zhang et al., (2012. Virology Journal 2012: 9:85) A recombinant Marek s disease virus that expresses the S1 gene from the QX like IBV inserted into the genome of the CVI1988/Rispens strain of MDV. SPF chickens that were vaccinated at day old with rmdv S1 found to have a low level of antibody response until 4 weeks post vaccination, but after IBV challenge at 4 weeks of age, the antibody level increased more rapidly compared with the control group. The chickens were protected when challenged with the QX like IBV 18
Zhang et al., 2014 (Vaccine 32:664 670) produced a modified baculovirus mediating transgene expressing the S1 glycoprotein of M41 strain on the baculovirus envelope under the control of a mammalian promoter, designated BacMam virus. 19
This product elicited stronger humoral and cell mediated immune responses and showed a greater capacity to induce cytotoxic T lymphocyte responses. Using this product as a vaccine, it resulted in 83% protection following challenge with IBV M41. 20
Lv et al., 2014. (J. Vet. Sci. 15:209) developed a chimeric virus like particle (VLP). The VLP was composed of matrix protein from avian influenza (H5N1) and fusion protein neuraminidase/spike protein S1. They immunized i.m. mice and chicken The chimeric VLPs induced significantly higher neutralization antibody level than inactivated H120 virus in SPF chickens. 21
Liu et al 2013 (vaccine 31:5524-5530) inserted M and S proteins to Baculovirus in Sf9 cells and assembled IBV VLPs. The generated VLPs induced, 28 days post vaccination humoral immune responses in a level comparable to that of inactivated IBV vaccine and elicited significantly higher cellular immune responses (Elispot of interferon and IL-4 secreting spleen cells) than the inactivated vaccine. 22
Falchieri et al., 2013 (Vaccine 31:2565 2571) Expressed either S1 or N genes of IBV in subtype A of AMPV. A bivalent recombinant was also prepared Day old chicks were vaccinated by eye drop, induced protection against virulent IBV QX challenge 3 weeks later, as assessed by tracheal cilia activity. Nonetheless no sero conversion nor major recombinant tracheal replication (by real time RT PCR) in the rampv/ibv vaccinated chicks could be observed. 23
Toro et al., 2014 (Avian Diseases 58:211) studied the protective properties of S1 proteins of 3 distinct ArkDPI vaccine subpopulations, expressed in a recombinant adenovirus vector which become selected in chickens after vaccination, and designated as C2, C4 and C5. Population C4 showed high prevalence after vaccination, C5 is rarely seen 24
best protection was conferred by S1.C2 followed by S1.C5. Chicken vaccinated with S1.C4 were indistinguishable from unvaccinated / challenged control chickens. These results were also confirmed by the differences in tears viral load, IBV specific IgG and IgA lymphocytes responses. The AA sequences are responsible to these differences 25
S2 protein. S2 remains largely unexposed to the immune system during natural infection. Speculatively overexposure to S2 likely induce an increase in B and T cells bearing receptors interacting with epitopes on the S2. 26
Toro et al., 2014 (Avian Diseases 58:83 89) developed Newcastle disease virus (LaSota) expressing IBV S2 gene. IBV heterotypic protection was assessed using a prime boost approach with a commercial attenuated Mass type vaccine. Their results demonstrated that priming with IBV S2, overexposing the chicken immune system with S2, followed by boost with whole virus protects chickens against IBV. Regarding incidence of detection of challenge IBV RNA in the trachea, viral RNA was detected in 50% of rls/ibv.s2 + Mass vaccination while chicken vaccinated twice with Mass or unvaccinated challenged control showed 84% and 90% incidence of IBV RNA detection COST FA1207 in Budapest the meeting trachea. 27
N protein The corona virus nucleocapsid (N) protein, an virus internal protein, plays a multifunctional role in the virus life cycle, from regulation of replication and transcription and genome packaging to modulation of host processes. 28
The N protein is expressed abundantly during infections. Thus it is a target protein when designing infectious bronchitis vaccine, and frequent target of diagnostic applications. Fang Yan et al., 2013 J. Vet. Sci. 14:53 60) Chickens immunized with pvax1 16N had a significant high level of IBV specific cellular proliferation and T lymphocytes 29
The N protein can induce high titers of crossreactive antibodies and cell mediated immunity, which protects chickens from acute infections. 30
M protein. The M protein is associated with virus assembly. The M glycoprotein can induce the production of detectable antibodies and delayed hypersensitivity responses 31
Fang Yan et al., 2013 (J. Vet. Sci. 14:53 60) constructed 4 plasmids (DNA ) expressing the S1, N and M proteins of a virulent IBV strain (pvax1 16S1, pvax1 16N, pvax1 16M and pvax1 16S1/M/N). Chickens were immunized monovalently with each individual plasmid or multivalent with combination of the three different plasmids, followed by boosting with an inactivated IBV before being challenged with virulent IBV. All Three IBV proteins have their own unique and important role in eliciting IBV immune responses. 32
Vaccination with pvax1 16M alone provoke the weakest immune response demonstrated by the lowest antibody titers, pvax1 16N moderate, and pvax1 16S1 the highest. Chickens immunized with pvax1 16N had a significant higher level of IBV specific cellular proliferation and T lymphocytes than birds immunized with pvax1 16M or pvax1 16S1. The challenge assay also proved that the DNA vaccine targeting the N protein is more effective. 33
Combination of S1, M and N genes provide stronger protection against IBV. Administration of the DNA vaccine resulted in effective cellular immunization and promote virus clearance. 34
Conclusions. S1 is the most important IBV protein, producing moderate humoral immune response but a good protection. S2 protein play some role in the humoral immune response to IBV The role of the N protein should not be overlooked as it stimulate cell mediated response. The role of the M protein is not well defined. 35
Chickens that received a combination of the three viral proteins mounted a stronger immune responses than the birds immunized with each of the proteins alone. 36
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