Immunoinformatics Approach for Multiepitopes Vaccine Prediction against Glycoprotein B of Avian Infectious Laryngotracheitis Virus.

Avian infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes an economically important respiratory chicken disease. The disease mainly controlled by vaccination. However conventional vaccinations increased the spread of the virus by latency. Therefore the aim of this study was to design multi epitopes vaccine against glycoprotein D of ILTV using immunoinformatics tools. The envelope glycoprotein D sequences were retrieved from the National Center for Biotechnology Information (NCBI) and aligned using Bioedit software for conservancy. The prediction of B and T cell epitopes were performed using Immune Epitope Database (IEDB) analysis resources. Homology modeling and docking were also performed to predict the binding affinity of the predicted epitopes to the chicken alleles. B cell prediction methods proposed nineteen linear epitopes, among them twelve epitopes were on surface and eleven antigenic epitopes using Bepipred, Emini surface accessibility and kolaskar antigenicity methods, respectively. However, only seven epitopes fulfilled the B cell prediction methods. Among these seven epitopes, two epitopes namely 256PRPDSVPQEIPAVTKK271 and 226 RHADDVY 232 were proposed as the top B cell epitopes. For T cells, three epitopes namely 24STAAVTYDY32, 20FASQSTAAV28 and 353FAAFVACAV361 were proposed as cytotoxic T cells (CTL) epitopes due to their great allele’s linkage to MHC class I alleles. MHC class II alleles extensively interacted with multiple epitopes. The best predicted epitopes were 88FEASVVWFY96, 212FQGEHLYPI220, 353FAAFVACAV361 and 137VDYVPSTLV145. Moreover, molecular docking revealed high binding affinity between chicken MHCI BF alleles and MHC1 docked epitopes (20FASQSTAAV28, 24STAAVTYDY32 and 353FAAFVACAV361) that indicated by the lower global energy scores. The In-silico analysis of ILTV glycoprotein D in this study suggested eight epitopes that could be a better choice as worldwide multi epitopes vaccine. These epitopes may effectively elicit both humoral and cell-mediated immunity. Furthermore in vitro and in vivo studies are required to support the effectiveness of these epitopes as vaccine candidates.

Background Small ruminant morbillivirus or peste des petits ruminants virus (PPRV) is an acute and highly contagious viral disease of goats, sheep, and other livestock. This study aimed at predicting an effective multiepitope vaccine against PPRV from the immunogenic proteins haemagglutinin (H), matrix (M), fusion (F), and nucleoprotein (N) using immunoinformatics tools. Materials and Methods The sequences of the immunogenic proteins were retrieved from GenBank of the National Center for Biotechnology Information (NCBI). BioEdit software was used to align each protein from the retrieved sequences for conservancy. Immune Epitope Database (IEDB) analysis resources were used to predict B and T cell epitopes. For B cells, the criteria for electing epitopes depend on the epitope linearity, surface accessibility, and antigenicity. Results Nine epitopes from the H protein, eight epitopes from the M protein, and ten epitopes from each of the F and N proteins were predicted as linear epitopes. The surface accessibility method proposed seven surface epitopes from each of the H and F proteins in addition to six and four epitopes from the M and N proteins, respectively. For antigenicity, only two epitopes 142PPERV146 and 63DPLSP67 were predicted as antigenic from H and M, respectively. For T cells, MHC-I binding prediction tools showed multiple epitopes that interacted strongly with BoLA alleles. For instance, the epitope 45MFLSLIGLL53 from the H protein interacted with four BoLA alleles, while 276FKKILCYPL284 predicted from the M protein interacted with two alleles. Although F and N proteins demonstrated no favorable interaction with B cells, they strongly interacted with T cells. For instance, 358STKSCARTL366 from the F protein interacted with five alleles, followed by 340SQNALYPMS348 and 442IDLGPAISL450 that interacted with three alleles each. The epitopes from the N protein displayed strong interaction with BoLA alleles such as 490RSAEALFRL498 that interacted with five alleles, followed by two epitopes 2ATLLKSLAL10 and 304QQLGEVAPY312 that interacted with four alleles each. In addition to that, four epitopes 3TLLKSLALF11, 356YFDPAYFRL364, 360AYFRLGQEM368, and 412PRQAQVSFL420 interacted with three alleles each. Conclusion Fourteen epitopes were predicted as promising vaccine candidates against PPRV from four immunogenic proteins. These epitopes should be validated experimentally through in vitro and in vivo studies.

Comparison of the effects of infectious bronchitis and infectious laryngotracheitis on the chicken respiratory tract

Mature laying chickens were inoculated intratracheally with a field strain of infectious laryngotracheitis (ILT) virus. Tracheal swabs were collected regularly from all birds for virus culture. At various times post-inoculation, pairs of birds were killed and tissues removed for detection of virus products using conventional tissue homogenization and culture, organ culture, indirect immunofluorescence (IF) and also the polymerase chain reaction (PCR). The latter was used to detect a DNA sequence from the ILT virus thymidine kinase gene. Following inoculation the birds developed mild respiratory disease with clinical signs characteristic of ILT from 3 to 10 days post-inoculation. Trachea and turbinate tissues were virus-positive as determined by virus isolation, organ culture, IF and PCR on day 4 post-inoculation. After recovery from the acute phase, virus shedding initially ceased, then intermittent, low level shedding was recorded for five of the six remaining birds. In an attempt to locate sites of latency, pairs of birds were sampled at 31, 46 and 61 days post-inoculation. Virus was not detected in upper respiratory tract or ocular tissues by conventional techniques, or in the trigeminal, proximal and distal ganglia. All tissues were also negative by PCR, except for the trigeminal ganglia of five of the six birds. All PCR-positive birds had previously shed ILT virus intermittently between days 19 and 59 post-inoculation. As we did not detect viral DNA in any of the other tissues sampled from clinically recovered birds, we conclude that the trigeminal ganglion is the main site of latency of ILT virus.

Development and application of a combined molecular and tissue culture-based approach to detect latent infectious laryngotracheitis virus (ILTV) in chickens

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that infects chickens, causing upper respiratory tract disease and significant losses to poultry industries worldwide. Glycoprotein G (gG) is a broad-range viral chemokine-binding protein conserved among most alphaherpesviruses, including ILTV. A number of studies comparing the immunological parameters between infection with gG-expressing and gG-deficient ILTV strains have demonstrated that expression of gG is associated with increased virulence, modification of the amount and the composition of the inflammatory response, and modulation of the immune responses toward antibody production and away from cell-mediated immune responses. The aims of the current study were to examine the establishment of infection and inflammation by ILTV and determine how gG influences that response to infection. In vitro infection studies using tracheal organ tissue specimen cultures and blood-derived monocytes and in vivo infection studies in specific-pathogen-free chickens showed that leukocyte recruitment to the site of infection is an important component of the induced pathology and that this is influenced by the expression of ILTV gG and changes in the transcription of the chicken orthologues of mammalian CXC chemokine ligand 8 (CXCL8), chicken CXCLi1 and chicken CXCLi2, among other cytokines and chemokines. The results from this study demonstrate that ILTV gG interferes with chemokine and cytokine transcription at different steps of the inflammatory cascade, thus altering inflammation, virulence, and the balance of the immune response to infection.IMPORTANCE Infectious laryngotracheitis virus is an alphaherpesvirus that expresses gG, a conserved broad-range viral chemokine-binding protein known to interfere with host immune responses. However, little is known about how gG modifies virulence and influences the inflammatory signaling cascade associated with infection. Here, data from in vitro and in vivo infection studies are presented. These data show that gG has a direct impact on the transcription of cytokines and chemokine ligands in vitro (such as chicken CXCL8 orthologues, among others), which explains the altered balance of the inflammatory response that is associated with gG during ILTV infection of the upper respiratory tract of chickens. This is the first report to associate gG with the dysregulation of cytokine transcription at different stages of the inflammatory cascade triggered by ILTV infection of the natural host.

Blue Tongue Disease (BTD) is a non-contagious insect transmitted disease of ruminants caused by double stranded RNA virus. This study aimed to predict an effective multi-epitopes vaccine against BTD from VP5 and VP7 as immunogenic proteins using immunoinformatic tools. The VP5 and VP7 proteins sequences were retrieved from GenBank of National Center for Biotechnology Information (NCBI). The sequences of each protein were aligned for conservancy using Bioedit software. Immune Epitope Database (IEDB) analysis resources were used to predict B and T cell epitopes. The proposed MHC-1 epitopes of both proteins were further subjected to molecular docking to show minimum binding energy of each epitopes. In our results, two epitopes (235-SEEV-235 and 85-PDPLSP-90) from VP5 and two epitopes (79-PISPDYTQ-86 and 297-PIFPPN-302) from VP7 were proposed as B cell epitopes since they were shown to be linear, surface accessible and antigenic epitopes. For T cells, MHC-1 binding prediction tools showed multiple epitopes strongly interacted with BoLA alleles from both VP5 and VP7. Among them three epitopes, (257-KLKKVINAL-265, 487-QMHILRGPL-495 and 350-VMMRFKIPR-358) fromVP5 protein and four epitopes (86-QHMATIGVL-94, 315-TLADVYTVL-323, 17-TLQEARIVL-25 and 10-TVMRACATL-18) from VP7 protein interacted with the highest number of alleles and demonstrated best binding affinity to MHC-1 alleles. Thus were proposed as a vaccine candidate from VP5 and VP7 proteins. All the epitopes from VP5 and VP7 that interacted with MHC-1 alleles when subjected to molecular docking against the sheep b_microglobulin alleles demonstrated biologically significant higher binding affinity which expressed by their lower global and attractive energy. In conclusion, eleven epitopes were predicted as promising vaccine candidates against BTD from the VP5 and VP7 immunogenic proteins. These epitopes require to be validated experimentally through in vitro and in vivo studies.

Infectious laryngotracheitis (ILT) is a highly contagious disease of the upper respiratory tract in chickens. The Gallid alpha herpesvirus type 1, also commonly known as ILT virus (ILTV), causes ILT. Although ILT was first reported in the United States in 1925, it has also been reported in other countries/regions of the world, including Australia, Asia, and Europe. The outcome of infection can be influenced by several factors, including the host’s age, exposure route, pathogenicity of the challenging virus, and initial viral load. In infected chickens, rough lesions are detected on the conjunctiva and throughout the respiratory system, but they are most commonly seen in the trachea and larynx. Other typical signs of the illness in poultry birds are expectoration of bloody mucus, severe dyspnea, coughing, gasping, and rales. Avian cell lines and embryodized chicken eggs are commonly used to isolate ILTV. Three types of ILT are associated with this infection: acute, chronic, and peracute. Chickens contract ILTV through the eyes and upper respiratory tracts. The main pathway by which ILTV is spread in poultry is through direct or indirect contact with sick/infected poultry birds, such as chickens and turkeys. No medication has been proven to be successful in curtailing clinical signs or lesion severity. Vaccination can prevent ILTV infection. Several biological and ecological characteristics of ILTV make its eradication from intensive poultry production locations quite likely. ILT is deemed a serious concern for poultry health, including its significant economic impact on the poultry industry. Therefore, this review highlights important comprehensive information regarding the impact of ILT in poultry, a major source of protein. In addition, a deeper understanding of the causes, signs, diagnosis, treatment, and prevention of ILT in poultry birds was comprehensively discussed.

Respiratory syncytial virus (RSV) is a major cause of serious acute lower respiratory tract infection in infants and the elderly. The lack of a licensed RSV vaccine calls for the development of vaccines with other targets and vaccination strategies. Here, we construct a recombinant protein, designated P-KFD1, comprising RSV phosphoprotein (P) and the E.-coli-K12-strain-derived flagellin variant KFD1. Intranasal immunization with P-KFD1 inhibits RSV replication in the upper and lower respiratory tract and protects mice against lung disease without vaccine-enhanced disease (VED). The P-specific CD4+ Tcells provoked by P-KFD1 intranasal (i.n.) immunization either reside in or migrate to the respiratory tract and mediate protection against RSV infection. Single-cell RNA sequencing (scRNA-seq) and carboxyfluorescein succinimidyl ester (CFSE)-labeled cell transfer further characterize the Th1 and Th17 responses induced by P-KFD1. Finally, we find that anti-viral protection depends on either interferon-γ (IFN-γ) or interleukin-17A (IL-17A). Collectively, P-KFD1 is a promising safe and effective mucosal vaccine candidate for the prevention of RSV infection.

Development of a Recombinant Infectious Bronchitis Virus Vaccine Expressing Infectious Laryngotracheitis Virus Multiple Epitopes

Infectious laryngotracheitis (ILT) is a respiratory disease that affects chickens. It is caused by the alphaherpesvirus, infectious laryngotracheitis virus (ILTV). This virus undergoes lytic replication in the epithelial cells of the trachea and upper respiratory tract (URT) and establishes latent infection in the trigeminal ganglia (TG) and trachea. Live attenuated vaccines are widely used to control ILT. At least one of these vaccines can establish latent infections in chickens, but this has not been demonstrated for all vaccines. The aim of the current study was to determine the capacity of three commercially available vaccines (SA2, A20 and Serva) and a glycoprotein G deletion mutant vaccine candidate (ΔgG ILTV) to establish latent infection in the TG of specific pathogen free (SPF) chickens. Five groups of 7-day-old SPF chickens were eye-drop vaccinated with either one of the vaccine strains or mock-vaccinated with sterile media and followed until 20 or 21 days post-vaccination (dpv). ILTV DNA was detected at 20–21 dpv in the TG of 23/40 (57.5%) vaccinated SPF chickens (SA2 = 10/10; A20 = 6/10; Serva = 3/10; ΔgG = 4/10) by PCR, but virus could not be reactivated from TG co-cultivated with primary chicken embryo kidney cells. In the birds from which ILTV DNA was detected in the TG, ILTV DNA could not be detected in the URT or trachea of 3 birds in each of the SA2, A20 and Serva vaccinated groups, and in 4 birds in the ΔgG vaccinated group, indicating that these birds were latently infected in the absence of active lytic replication and virus shedding. Results from this study demonstrate the capacity of commercial ILTV vaccines to establish latent infections and underline their importance in the epidemiology of this disease.

Evaluation of immunological responses to a glycoprotein G deficient candidate vaccine strain of infectious laryngotracheitis virus

Rift Valley fever virus (RVFV) is a single strand, negative sense, an envelope spherical particle, of size 80 - 120 nm, segmented RNA virus that belongs to Genus: Phlebovirus of Bunyaviridae family. The clinical manifestations of the disease among animals are abortion and death of newborns. While in humans, although the disease is mild or asymptomatic, there are several reports of high fatality rates. The M segment of RVF virus Genome which encodes the envelope glycoprotein has been used to design a vaccine for immunization against this virus. we aimed to design a novel peptide-based vaccine for RVFV using immunoinformatic approach to predict highly conserved epitopes against glycoprotein receptor Gn and Gc of M protein, that can mediate immune response which can use later to produce a new vaccine that could replace the conventional vaccine. A total of 118 sequences of M protein of RVFV were retrieved from NCBI database and stored as FASTA format for immunoinformatics analysis. ClustalW multiple alignment using BioEdit sequence alignment editor (v7.0.9) was performed to the retrieved sequences to identify the conserved region compared to M protein RVFV reference sequence under gene bank accession number [YP_003848705.1]. The B and T cell epitopes prediction is done by immune epitope database (IEDB). (IEDB) predicted B cell epitopes by Bepipred linear epitope prediction analysis and T cell epitopes using Major Histocompatibility Complex class I and ll binding prediction tool based on Stabilized Matrix Method (SMM). Allergenicity for the Helper T cell epitopes (HTL) predicted using AllerTop software. TAP transporter and Proteasomal cleavage for Cytotoxic T cell (CTL) were predicted from (IEDB). The population coverage over the world was determined. The four best predicted CTL namely (836HTYLQSVRK844, 672IPRYSTYLM680, 1085ILHFTVPEV1093 and 834FVHTYLQSV842) were docked with HLA-B*35 and suggested to be universal peptide vaccine for immunization against RVFV. The typical overlapping between the MHC Class I epitope (834FVHTYLQSV842) and MHC Class II (834FVHTYLQSV842) suggest the possibility to presenting these antigens to immune system via both MHC class I and II pathways. In conclusion; the four CTL epitopes are selected as vaccine candidates to develop safer and easier to manufacture without need of culture vaccine for prophylactic method against this virus. We recommend to confirm our result by doing additional in vivo and in vitro complementary steps to support this novel predicted vaccine.

Dengue is a viral mosquito-borne disease that rapidly spreads in tropical and subtropical countries, including the Philippines. One of its most distinguishing characteristics is the ability of the Dengue Virus (DENV) to easily surpass the innate responses of the body, thus activating B cells of the adaptive immunity to produce virus-specific antibodies. Moreover, Dengvaxia® is the only licensed vaccine for DENV, but recent studies showed that seronegative individuals become prone to increased disease severity and hospitalization. Owing to this limitation of the dengue vaccine, this study determined and compared consensus and unique B cell epitopes among each DENV (1–4) Philippine isolate to identify potential areas of interest for future vaccine studies and therapeutic developments. An in silico-based epitope prediction of forty (40) DENV 1–4 strains, each serotype represented by ten (10) sequences from The National Center for Biotechnology Information (NCBI), was conducted using Kolaskar and Tongaonkar antigenicity, Emini surface accessibility, and Parker hydrophilicity prediction in Immune Epitope Database (IEDB). Results showed that five (5) epitopes were consensus for DENV-1 with no detected unique epitope, one (1) consensus epitope for DENV-2 with two (2) unique epitopes, one (1) consensus epitope for DENV-3 plus two (2) unique epitopes, and two (2) consensus epitopes and one (1) unique epitope for DENV-4. The findings of this study would contribute to determining potential vaccine and diagnostic marker candidates for further research studies and immunological applications against DENV (1–4) Philippine isolates.

Previous studies in our laboratory using a combination of polymerase chain reaction and restriction fragment length polymorphism have identified at least five different genotypes of infectious laryngotracheitis virus (ILTV). However, the virulence of these classes of ILTV was not investigated. In this study, five groups (16 birds each) of 3-week-old specific pathogen free chickens were inoculated via the intratracheal route with 10(3) median embryo infected dose of five different strains of ILTV. Three further groups of chickens were inoculated similarly with the vaccine strains SA2 and A20 or with sterile phosphate-buffered saline (PBS) for comparison. Four days post-inoculation, clinical signs were monitored for scoring, and eight chickens from each group were subsequently euthanized, weighed and subjected to pathological and histopathological examinations. The remaining birds were monitored for clinical signs and mortality until 21 days post-inoculation. All groups inoculated with ILTV strains showed moderate to severe clinical signs 4 days after inoculation. The strain Q1-96 caused only minimal breathing symptoms with a median score that was not significantly different to that of the group inoculated with PBS, but was significantly different to those of the groups inoculated with other ILTV strains. The strain Q1-96 caused severe photophobia and conjunctivitis with a median score that was significantly higher than those of all other groups except for the group inoculated with the strain N3-04. All ILTV strains caused a significant reduction in weight gain when compared with the group inoculated with PBS. The strain Q1-96 caused an average weigh loss of 14% that was significantly higher than those of other ILTV strains. The strains S2-04 and Q1-96 induced only minor microscopic tracheal lesions while all the other ILTV strains, including the vaccine strains A20 and SA2, induced moderate to severe microscopic tracheal lesions. Median scores for microscopic tracheal lesions were well correlated with the number of viral genomes detected in trachea. The results revealed that there is considerable variation among ILTV strains in their tropism for trachea or conjunctiva. In addition it was revealed that ILTV strains with high affinity for conjunctiva can severely affect weigh gain. The ILTV numbers and microscopic lesions in trachea were not found to be reliable indicators of virulence since they are not necessarily correlated with mortality rate in ILT.