Search Results (27)

Background/Objectives: More than 33 billion chickens are industrially raised for meat and egg production globally and vaccinated against Marek’s disease virus (MDV). The antigenically related herpesvirus of turkey (HVT) is used as a live-attenuated vaccine, commonly provided as a recombinant vector to protect chickens against additional unrelated pathogens. Because HVT replicates in a strictly cell-associated fashion to low levels of infectious units, adherent primary chicken or duck embryo fibroblasts are infected, dislodged from the cultivation surface and distributed as cryocultures in liquid nitrogen to the site of application. Although viable cells are complex products, application of infected cells in ovo confers protection even in presence of maternal antibodies. Methods/Results: The aim of our study was to determine whether a continuous cell line in a scalable cultivation format can be used for production of HVT-based vaccines. The AGE1.CR cell line (from Muscovy duck) was found to be highly permissive in adherent cultures. Propagation in suspension, however, initially gave very low yields. The induction of cell-to-cell contacts in carrier-independent suspensions and a metabolic shock improved titers to levels suitable for vaccine production (>10⁵ infectious units/mL after infection with multiplicity of 0.001). Conclusions: Production of HVT is challenging to scale to large volumes and the reliance on embryonated eggs from biosecure facilities is complex. We demonstrate that a cell-associated HVT vector can be propagated in a carrier-independent suspension culture of AGE1.CR cells in chemically defined medium. The fed-batch production is independent of primary cells and animal-derived material and can be scaled to large volumes. Full article

The glycoprotein C (gC) of gallid alphaherpesvirus 2—better known as Marek’s disease (MD) virus (MDV)—and gallid alphaherpesvirus 3 is required for horizontal transmission in chickens. Since gC is conserved within the Alphaherpesvirinae subfamily, we hypothesized that gC was also essential for the horizontal transmission of meleagrid alphaherpesvirus 1 (MeAHV1) or turkey herpesvirus (HVT). To test this hypothesis, we generated a fluorescent protein-tagged clone of recombinant (r)HVT (vHVT47G), removed the open reading frame of HVT gC from the genome (vHΔgC), and rescued the deletion by inserting an HA-epitope tagged HVT gC (vHΔgC-R) to test their ability to transmit in chickens and turkeys. We also tested whether MDV gC could compensate for HVT gC during transmission, where HVT gC was replaced with MDV gC (vH-MDVgC). Although all viruses replicated in chickens, none spread from chicken to chicken. However, when tested in turkeys, all viruses except vHΔgC transmitted from turkey to turkey. Importantly, the rescuent virus (vHΔgC-R) and HVT expressing MDV gC (vH-MDVgC) rescued transmission, showing that HVT gC is required and MDV gC can compensate for HVT gC for turkey-to-turkey transmission. These data confirm the host-specific transmission of HVT in turkeys and suggest that the essential function of alphaherpesvirus gC proteins is conserved. This information can be exploited while generating future vaccines against MD that will affect the poultry industry worldwide. Full article

Herpesvirus of turkey (HVT) recombinant vector vaccines are widely used in the poultry industry. However, due to limitations in loading multiple foreign antigens into a single HVT vector, other viral vectors are urgently needed. Since chickens lack maternal immunity to duck enteritis virus (DEV), vector vaccines using DEV as a backbone are currently under study. Even though a recently developed DEV vector vaccine expressing the influenza hemagglutinin H5 of highly pathogenic avian influenza (DEV-H5) induces highly detectable anti-HA antibodies, safety issues hamper further vaccine development. In this work, tissue affinity and horizontal transmission in 1-day-old chickens were systematically evaluated after DEV-H5 vector vaccine inoculation. Sixty percent of DEV-H5-inoculated chickens died between day 2 and day 7 post-inoculation. The displayed clinical signs consisted of lethargy, anorexia, and diarrhea, and virus was shed in feces. Gross and/or histological lesions were recorded in the kidney, heart, intestine, liver, lung, and spleen. Moreover, DEV-H5 replication in intestinal cells caused an increment in interferon-α expression, while occluding junction proteins and ZO-1 expression were significantly upregulated. As a control, birds inoculated with a commercial recombinant turkey herpesvirus expressing the VP2 protein of the infectious bursal disease virus (HVT-VP2) vector vaccine showed neither clinical signs nor mortality. Overall, while the HVT-VP2 vaccine demonstrated complete safety in 1-day-old chickens, our potential DEV-H5 vaccine requires further attenuation for consideration as a vector vaccine candidate in chickens. Full article

Inactivated and live attenuated vaccines are the mainstays of preventing viral poultry diseases. However, the development of recombinant DNA technology in recent years has enabled the generation of recombinant virus vector vaccines, which have the advantages of preventing multiple diseases simultaneously and simplifying the vaccination schedule. More importantly, some can induce a protective immune response in the presence of maternal antibodies and offer long-term immune protection. These advantages compensate for the shortcomings of traditional vaccines. This review describes the construction and characterization of primarily poultry vaccine vectors, including fowl poxvirus (FPV), fowl adenovirus (FAdV), Newcastle disease virus (NDV), Marek’s disease virus (MDV), and herpesvirus of turkey (HVT). In addition, the pathogens targeted and the immunoprotective effect of different poultry recombinant virus vector vaccines are also presented. Finally, this review discusses the challenges in developing vector vaccines and proposes strategies for improving immune efficacy. Full article

Newcastle disease (ND) is a significant infectious disease in poultry, causing substantial economic losses in developing countries. To control ND, chickens must be vaccinated multiple times a year. In order to develop an improved vaccine that provides long-term protection, the F gene from genotype VII NDV was inserted into the herpesvirus of turkey (HVT) vaccine virus using CRISPR/Cas9-mediated NHEJ repair and Cre/LoxP technology. The immunogenicity and protective efficacy of the resulting recombinant vaccines were evaluated through antibody assays and virus challenge experiments. Two recombinant vaccines, rHVT-005/006-F and rHVT-US2-F, were generated, both exhibiting growth rates comparable with those of HVT in vitro and consistently expressing the F protein. One-day-old specific pathogen-free (SPF) chickens immunized with 2000 PFU/bird of either rHVT-005/006-F or rHVT-US2-F developed robust humoral immunity and were completely protected against challenge with the NDV F48E8 strain at 4 weeks post-vaccination (wpv). Furthermore, a single dose of these vaccines provided sustained protection for at least 52 wpv. Our study identifies rHVT-005/006-F and rHVT-US2-F as promising ND vaccine candidates, offering long-term protection with a single administration. Moreover, HVT-005/006 demonstrates promise for accommodating additional foreign genes, facilitating the construction of multiplex vaccines. Full article

Marek’s disease (MD), caused by gallid alphaherpesvirus 2 (GaAHV2) or Marek’s disease herpesvirus (MDV), is a devastating disease in chickens characterized by the development of lymphomas throughout the body. Vaccine strains used against MD include gallid alphaherpesvirus 3 (GaAHV3), a non-oncogenic chicken alphaherpesvirus homologous to MDV, and homologous meleagrid alphaherpesvirus 1 (MeAHV1) or turkey herpesvirus (HVT). Previous work has shown most of the MDV gC produced during in vitro passage is secreted into the media of infected cells although the predicted protein contains a transmembrane domain. We formerly identified two alternatively spliced gC mRNAs that are secreted during MDV replication in vitro, termed gC104 and gC145 based on the size of the intron removed for each UL44 (gC) transcript. Since gC is conserved within the Alphaherpesvirinae subfamily, we hypothesized GaAHV3 (strain 301B/1) and HVT also secrete gC due to mRNA splicing. To address this, we collected media from 301B/1- and HVT-infected cell cultures and used Western blot analyses and determined that both 301B/1 and HVT produced secreted gC. Next, we extracted RNAs from 301B/1- and HVT-infected cell cultures and chicken feather follicle epithelial (FFE) skin cells. RT-PCR analyses confirmed one splicing variant for 301B/1 gC (gC104) and two variants for HVT gC (gC104 and gC145). Interestingly, the splicing between all three viruses was remarkably conserved. Further analysis of predicted and validated mRNA splicing donor, branch point (BP), and acceptor sites suggested single nucleotide polymorphisms (SNPs) within the 301B/1 UL44 transcript sequence resulted in no gC145 being produced. However, modification of the 301B/1 gC145 donor, BP, and acceptor sites to the MDV UL44 sequences did not result in gC145 mRNA splice variant, suggesting mRNA splicing is more complex than originally hypothesized. In all, our results show that mRNA splicing of avian herpesviruses is conserved and this information may be important in developing the next generation of MD vaccines or therapies to block transmission. Full article

Since the outbreak of the H9N2/Y439 avian influenza virus in 1996, the Korean poultry industry has incurred severe economic losses. A novel possibly zoonotic H9N2 virus from the Y280-like lineage (H9N2/Y280) has been prevalent in Korea since June 2020, posing a threat to the poultry sector. Rapid mutation of influenza viruses urges the development of effective vaccines against newly generated strains. Thus, we engineered a recombinant virus rHVT/Y280 to combat H9N2/Y280. We integrated the hemagglutinin (HA) gene of the H9N2/Y280 strain into the US2 region of the herpesvirus of turkeys (HVT) Fc126 vaccine strain, utilizing CRISPR/Cas9 gene-editing technology. The successful construction of rHVT/Y280 was confirmed by polymerase chain reaction and sequencing, followed by efficacy evaluation. Four-day-old specific pathogen-free chickens received the rHVT/Y280 vaccine and were challenged with the H9N2/Y280 strain A21-MRA-003 at 3 weeks post-vaccination. In 5 days, there were no gross lesions among the vaccinated chickens. The rHVT/Y280 vaccine induced strong humoral immunity and markedly reduced virus shedding, achieving 100% inhibition of virus recovery in the cecal tonsil and significantly lowering tissue viral load. Thus, HVT vector vaccines expressing HA can be used for protecting poultry against H9N2/Y280. The induction of humoral immunity by live vaccines is vital in such cases. In summary, the recombinant virus rHVT/Y280 is a promising vaccine candidate for the protection of chickens against the H9N2/Y280. Full article

Infectious bursal disease (IBD), caused by IBD virus (IBDV), is an extremely contagious immunosuppressive disease that causes major losses for the poultry industry worldwide. Recently, the novel variant IBDV (G2d) has been highly prevalent in Korea, but the current vaccines against this very virulent IBDV have limited efficacy against this novel variant. To develop a vaccine against this variant IBDV, a recombinant virus designated rHVT-VP2 was constructed by inserting the IBDV (G2d) VP2 gene into herpesvirus of turkeys (HVT) using CRISPR/Cas9 gene-editing technology. The PCR and sequencing results obtained showed that the recombinant virus rHVT-VP2 was successfully constructed. Vaccination with rHVT-VP2 generated IBDV-specific antibodies in specific pathogen-free chickens starting from 2 weeks post-immunization. Seven days after the challenge, the autopsy results showed that the bursa atrophy rates of the rHVT-VP2, HVT, vaccine A, and positive control groups were 0%, 100%, 60%, and 100%, respectively, and the BBIX values were 1.07 ± 0.22, 0.27 ± 0.05, 0.64 ± 0.33, and 0.32 ± 0.06, respectively. These results indicate that rHVT-VP2 can provide 100% protection against a challenge with the IBDV (G2d), whereas vaccine A only provides partial protection. In conclusion, vaccination with the recombinant virus rHVT-VP2 can provide chickens with effective protection against variant IBDV (G2d). Full article

Infectious laryngotracheitis (ILT) is an economically important disease in chickens. We previously showed that an in ovo adjuvantation of recombinant herpesvirus of the turkey-Laryngotracheitis (rHVT-LT) vaccine with CpG-oligonucleotides (ODN) can boost vaccine-induced responses in one-day-old broiler chickens. Here, we evaluated the protective efficacy of in ovo administered rHVT-LT + CpG-ODN vaccination against a wild-type ILT virus (ILTV) challenge at 28 days of age and assessed splenic immune gene expression as well as cellular responses. A chicken-embryo-origin (CEO)-ILT vaccine administered in water at 14 days of age was also used as a comparative control for the protection assessment. The results showed that the rHVT-LT + CpG-ODN or the CEO vaccinations provided significant protection against the ILTV challenge and that the level of protection induced by both the vaccines was statistically similar. The protected birds had a significantly upregulated expression of interferon (IFN)γ or interleukin (IL)-12 cytokine genes. Furthermore, the chickens vaccinated with the rHVT-LT + CpG-ODN or CEO vaccine had a significantly higher frequency of γδ T cells and activated CD4+ or CD8+ T cells, compared to the unvaccinated-ILTV challenge control. Collectively, our findings suggest that CpG-ODN can be used as an effective adjuvant for rHVT-LT in ovo vaccination to induce protective immunity against ILT in broiler chickens. Full article

Fowl cholera is caused by the bacterium Pasteurella multocida, a highly transmissible avian ailment with significant global implications, leading to substantial economic repercussions. The control of fowl cholera outbreaks primarily relies on vaccination using traditional vaccines that are still in use today despite their many limitations. In this research, we describe the development of a genetically engineered herpesvirus of turkeys (HVT) that carries the OmpH gene from P. multocida integrated into UL 45/46 intergenic region using CRISPR/Cas9-NHEJ and Cre-Lox system editing. The integration and expression of the foreign cassettes were confirmed using polymerase chain reaction (PCR), indirect immunofluorescence assays, and Western blot assays. The novel recombinant virus (rHVT-OmpH) demonstrated stable integration of the OmpH gene even after 15 consecutive in vitro passages, along with similar in vitro growth kinetics as the parent HVT virus. The protective efficacy of the rHVT-OmpH vaccine was evaluated in vaccinated ducks by examining the levels of P. multocida OmpH-specific antibodies in serum samples using ELISA. Groups of ducks that received the rHVT-OmpH vaccine or the rOmpH protein with Montanide™ (SEPPIC, Paris, France) adjuvant exhibited high levels of antibodies, in contrast to the negative control groups that received the parental HVT or PBS. The recombinant rHVT-OmpH vaccine also provided complete protection against exposure to virulent P. multocida X-73 seven days post-vaccination. This outcome not only demonstrates that the HVT vector possesses many characteristics of an ideal recombinant viral vaccine vector for protecting non-chicken hosts, such as ducks, but also represents significant research progress in identifying a modern, effective vaccine candidate for combatting ancient infectious diseases. Full article

Infectious laryngotracheitis (ILT) is an economically important disease of chickens. While the recombinant vaccines can reduce clinical disease severity, the associated drawbacks are poor immunogenicity and delayed onset of immunity. Here, we used CpG-oligonucleotides (ODN) as an in ovo adjuvant in boosting recombinant herpesvirus of turkey-laryngotracheitis (rHVT-LT) vaccine-induced responses in one-day-old broiler chickens. Two CpG-ODN doses (5 and 10 μg/egg) with no adverse effect on the vaccine-virus replication or chick hatchability were selected for immune-response evaluation. Results showed that while CpG-ODN adjuvantation induced an increased transcription of splenic IFNγ and IL-1β, and lung IFNγ genes, the IL-1β gene expression in the lung was significantly downregulated compared to the control. Additionally, the transcription of toll-like receptor (TLR)21 in the spleen and lung and inducible nitric oxide synthase (iNOS) in the spleen of all vaccinated groups was significantly reduced. Furthermore, splenic cellular immunophenotyping showed that the CpG-ODN-10μg adjuvanted vaccination induced a significantly higher number of macrophages, TCRγδ+, and CD4+ T cells as well as a higher frequency of activated T cells (CD4+CD44+) when compared to the control. Collectively, the findings suggested that CpG-ODN can boost rHVT-LT-induced immune responses in day-old chicks, which may help in anti-ILT defense during their later stages of life. Full article

Turkey herpesvirus (HVT) is widely used as an effective recombinant vaccine vector for expressing protective antigens of multiple avian pathogens from different loci of the HVT genome. These include the HVT029/031 (UL22–23) locus for the insertion of IBDV VP2 and the recently identified HVT005/006 locus as a novel site for expressing heterologous proteins. In order to compare the efficacy of recombinant vaccines with the HA gene at different sites, the growth curves and the HA expression levels of HVT-005/006-hCMV-HA, HVT-005/006-MLV-HA, and HVT-029/031-MLV-HA were first examined in vitro. While the growth kinetics of three recombinant viruses were not significantly different from those of parent HVT, higher expression of the HA gene was achieved from the HVT005/006 site than that from the HVT029/031 site. The efficacy of the three recombinant viruses against avian influenza H9N2 virus was also evaluated using one-day-old SPF chickens. Chickens immunized with HVT-005/006-MLV-HA or HVT-005/006-hCMV-HA displayed reduced virus shedding compared to HVT-029/031-MLV-HA vaccinated chickens. Moreover, the overall hemagglutination inhibition (HI) antibody titers of HVT-005/006-HA-vaccinated chickens were higher than that of HVT-029/031-HA-vaccinated chickens. However, HVT-005/006-MLV-HA and HVT-005/006-hCMV-HA did not result in a significant difference in the level of HA expression in vitro and provided the same protective efficacy (100%) at 5 days after challenge. In the current study, the results suggested that recombinant HVT005/006 vaccines caused better expression of HA than recombinant HVT029/031 vaccine, and that HVT-005/006-MLV-HA or HVT-005/006-hCMV-HA could be a candidate vaccine for the protection of chickens against H9N2 influenza. Full article

The increased virulence of infectious bursal disease virus (IBDV) is a threat to the chicken industry. The construction of novel herpesvirus of turkey-vectored (HVT) vaccines expressing VP2 of virulent IBDV may be a promising vaccine candidate for controlling this serious disease in chickens. We generated a novel infectious clone of HVT Fc-126 by inserting mini-F sequences in lieu of the glycoprotein C (gC) gene. Based on this bacterial artificial chromosome (BAC), a VP2 expression cassette containing the pMCMV IE promoter and a VP2 sequence from the virulent IBDV NJ09 strain was inserted into the noncoding area between the UL55 and UL56 genes to generate the HVT vector VP2 recombinant, named HVT-VP2-09. The recovered vectored mutant HVT-VP2-09 exhibited higher titers (p = 0.0202 at 36 h) or similar growth kinetics to the parental virus HVT Fc-126 (p = 0.1181 at 48 h and p = 0.1296 at 64 h). The high reactivation ability and strong expression of VP2 by HVT-VP2-09 in chicken embryo fibroblasts (CEFs) were confirmed by indirect immunofluorescence (IFA) and Western blotting. The AGP antibodies against IBDV were detected beginning at 3 weeks post-inoculation (P.I.) of HVT-VP2-09 in 1-day-old SPF chickens. Seven of ten chickens immunized with HVT-VP2-09 were protected post-challenge (P.C.) with the virulent IBDV NJ09 strain. In contrast, all chickens in the challenge control group showed typical IBD lesions in bursals, and eight of ten died P.C. In this study, we demonstrated that (i) a novel HVT BAC with the whole genome of the Fc-126 strain was obtained with the insertion of mini-F sequences in lieu of the gC gene; (ii) HVT-VP2-09 harboring the VP2 expression cassette from virulent IBDV exhibited in vitro growth properties similar to those of the parental HVT virus in CEF cells; and (iii) HVT-VP2-09 can provide efficient protection against the IBDV NJ09 strain. Full article

In this study, we developed a new recombinant virus rHVT-F using a Turkey herpesvirus (HVT) vector, expressing the fusion (F) protein of the genotype XII Newcastle disease virus (NDV) circulating in Peru. We evaluated the viral shedding and efficacy against the NDV genotype XII challenge in specific pathogen-free (SPF) chickens. The F protein expression cassette was inserted in the unique long (UL) UL45–UL46 intergenic locus of the HVT genome by utilizing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene-editing technology via a non-homologous end joining (NHEJ) repair pathway. The rHVT-F virus, which expressed the F protein stably in vitro and in vivo, showed similar growth kinetics to the wild-type HVT (wtHVT) virus. The F protein expression of the rHVT-F virus was detected by an indirect immunofluorescence assay (IFA), Western blotting, and a flow cytometry assay. The presence of an NDV-specific IgY antibody was detected in serum samples by an enzyme-linked immunosorbent assay (ELISA) in SPF chickens vaccinated with the rHVT-F virus. In the challenge experiment, the rHVT-F vaccine fully protects a high, and significantly reduced, virus shedding in oral at 5 days post-challenge (dpc). In conclusion, this new rHVT-F vaccine candidate is capable of fully protecting SPF chickens against the genotype XII challenge. Full article

In Alberta, infectious laryngotracheitis virus (ILTV) infection is endemic in backyard poultry flocks; however, outbreaks are only sporadically observed in commercial flocks. In addition to ILTV vaccine revertant strains, wild-type strains are among the most common causes of infectious laryngotracheitis (ILT). Given the surge in live attenuated vaccine-related outbreaks, the goal of this study was to assess the efficacy of a recombinant herpesvirus of turkey (rHVT-LT) vaccine against a genotype VI Canadian wild-type ILTV infection. One-day-old specific pathogen-free (SPF) White Leghorn chickens were vaccinated with the rHVT-LT vaccine or mock vaccinated. At three weeks of age, half of the vaccinated and the mock-vaccinated animals were challenged. Throughout the experiment, weights were recorded, and feather tips, cloacal and oropharyngeal swabs were collected for ILTV genome quantification. Blood was collected to isolate peripheral blood mononuclear cells (PBMC) and quantify CD4+ and CD8+ T cells. At 14 dpi, the chickens were euthanized, and respiratory tissues were collected to quantify genome loads and histological examination. Results showed that the vaccine failed to decrease the clinical signs at 6 days post-infection. However, it was able to significantly reduce ILTV shedding through the oropharyngeal route. Overall, rHVT-LT produced a partial protection against genotype VI ILTV infection. Full article