Recombinant Herpes Research Articles

Evaluation of vaccination against infectious laryngotracheitis (ILT) with recombinant herpesvirus of turkey (rHVT-LT) and chicken embryo origin (CEO) vaccines applied alone or in combination

ABSTRACTThe chicken embryo origin (CEO) infectious laryngotracheitis (ILT) live attenuated vaccines, although capable of protecting against disease and reducing challenge virus replication, can regain virulence. Recombinant ILT vaccines do not regain virulence but are partially successful at blocking challenge virus replication. The objective of this study was to evaluate the effect of rHVT-LT vaccination on CEO replication and how this vaccination strategy enhances protection and limits challenge virus transmission to naïve contact chickens. The rHVT-LT vaccine was administered at 1 day of age subcutaneously and the CEO vaccine was administered at 6 weeks of age via eye-drop or drinking water. CEO vaccine replication post vaccination, challenge virus replication and transmission post challenge were evaluated. After vaccination, only the group that received the CEO via eye-drop developed transient conjunctivitis. A significant decrease in CEO replication was detected for the rHVT-LT + CEO groups as compared to groups that received CEO alone. After challenge, reduction in clinical signs and challenge virus replication were observed in all vaccinated groups. However, among the vaccinated groups, the rHVT-LT group presented higher clinical signs and challenge virus replication. Transmission of the challenge virus to naïve contact chickens was only observed in the rHVT-LT vaccinated group of chickens. Overall, this study found that priming with rHVT-LT reduced CEO virus replication and the addition of a CEO vaccination provided a more robust protection than rHVT alone. Therefore, rHVT-LT + CEO vaccination strategy constitutes an alternative approach to gain better control of the disease.

A Recombinant Turkey Herpesvirus Expressing F and HN Genes of Avian Avulavirus-1 (AAvV-1) Genotype VI Confers Cross-Protection against Challenge with Virulent AAvV-1 Genotypes IV and VII in Chickens.

Newcastle disease (ND) is responsible for significant economic losses in the poultry industry. The disease is caused by virulent strains of Avian avulavirus 1 (AAvV-1), a species within the family Paramyxoviridae. We developed a recombinant construct based on the herpesvirus of turkeys (HVT) as a vector expressing two genes: F and HN (HVT-NDV-F-HN) derived from the AAvV-1 genotype VI (“pigeon variant” of AAvV-1). This recombinant viral vaccine candidate was used to subcutaneously immunize one group of specific pathogen-free (SPF) chickens and two groups of broiler chickens (20 one-day-old birds/group). Humoral immune response was evaluated by hemagglutination-inhibition test and enzyme-linked immunosorbent assay (ELISA). The efficacy of the immunization was assessed in two separate challenge studies performed at 6 weeks of age with the use of virulent AAvV-1 strains representing heterologous genotypes IV and VII. The developed vaccine candidate elicited complete protection in SPF chickens since none of the birds became sick or died during the 2-week observation period. In the broiler groups, 90% and 100% clinical protection were achieved after challenges with AAvV-1 of IV and VII genotypes, respectively. We found no obvious relationship between antibody levels and protection assessed in broilers in the challenge study. The developed recombinant HVT-NDV-F-HN construct containing genes from a genotype VI AAvV-1 offers promising results as a potential vaccine candidate against ND in chickens.

Abstract 1452: Development of ONCR-148, a miR-attenuated oncolytic HSV-1 designed to potently activate antitumor T cell response

Abstract Oncolytic viruses, through cancer cell-selective lysis and accompanying release of danger signals that promote immune activation, have demonstrated antitumor efficacy in monotherapy of metastatic melanoma and have shown promising activity in combination with checkpoint inhibitors. We present here the activity of ONCR-148, a recombinant oncolytic Herpes Simplex Virus (oHSV) designed to be a safe and efficacious therapy for the treatment of solid tumors. Neuron-specific miR attenuation of ICP4 gene inhibits viral replication in neurons while preserving its potent oncolytic activity in tumor cells. The antitumor potency of the virus is enhanced by incorporating in its genome a transgene expressing fibroblast activation protein (FAP)- and CD3- bispecific antibody that recruits and re-directs the cytolytic activity of T cells toward FAP-expressing stromal cells. FAP is a cell-surface serine protease that is almost exclusively found in fibroblasts within tumor stroma. However, systemic administration of a FAP-CD3 bispecific antibody was not tolerated due to the toxicity toward FAP-expressing bone marrow mesenchymal cells (Tran et al., 2013. J.Exp.Med, 210(6), 1125-1135). Thus, the local intra-tumoral delivery of FAP-CD3 is an attractive approach to promote tumor lysis and T cell activation against cancerous lesions. To test the in vivo activity of ONCR-148, as FAP is poorly expressed in syngeneic mouse tumor models, we developed a bilateral mouse FAP-expressing MC38 syngeneic colon carcinoma model (MC38-FAP), allowing to test local antitumor activity in oHSV-injected tumors and abscopal efficacy mediated by immune cells in the non-injected tumors. Intra-tumoral administration of ONCR-148 in the MC38-FAP model resulted in tumor growth inhibition of 60% (p<0.03) and 46% (p<0.01) on the injected (ipsilateral) and non-injected (contralateral) tumor, respectively. We then investigated additional transgenes that could potentiate the antitumor activity of ONCR-148. We combined in vivo ONCR-148 with ONCR-153, an oHSV armed with the NK and T cell activating cytokine IL-12 and the chemokines CXCL10 and CCL4, allowing for expansion and recruitment of T cells and antigen presenting cells in the tumor milieu. Combined intra-tumoral administration of ONCR-148 and ONCR-153 increased the antitumor response to 90% on the injected tumor (p<0.0001), leading to complete tumor remission in 5 out of 9 treated mice and to 80% on distant tumors (p<0.0001, with no complete responses observed).These data indicate that oHSV expressing bispecific antibodies, while being well tolerated, is efficacious in injected tumors and mediate systemic antitumor immune response. The efficacy can be potentiated by a local co-expression of immunostimulatory cytokines. Such data support the further development of oHSV-1 armed with CD3 bispecific antibodies for the therapy of patients with metastatic cancer. Citation Format: Agnieszka Denslow, Brian B. Haines, Michael S. Ball, Jacqueline Gursha, Daniel Wambua, Cecilia Kwong, Lingxin Kong, Allison Colthart, Prajna Behera, Peter Grzesik, Jennifer Lee, Terry Farkaly, Caitlin Goshert, Edward M. Kennedy, Lorena Lerner, Christophe Quéva. Development of ONCR-148, a miR-attenuated oncolytic HSV-1 designed to potently activate antitumor T cell response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1452.

Abstract 940: Development of ONCR-177, an miR-attenuated oncolytic HSV-1 designed to potently activate systemic antitumor immunity

Abstract ONCR-177 is a highly modified recombinant oncolytic Herpes Simplex Virus (oHSV) designed to be a safe and efficacious therapy for the treatment of solid tumor indications. Complementary safety mechanisms, such as tissue-specific miR attenuation of essential viral genes and UL37 mutation, were introduced to reduce replication, neuropathic activity, and latency in normal cells, while preserving oncolytic ability in tumor cells. In addition, ONCR-177 is armed with five transgenes: the NK and T cell activating cytokine IL-12, the chemokines CCL4 and FLT3LG (extracellular domain) to allow for expansion and recruitment of classical dendritic cells, and antagonists to the clinically validated immune checkpoint targets PD-1 and CTLA-4 to counter T cell exhaustion. Therefore, ONCR-177 is proposed to have a dual mechanism of action whereby the miR attenuation strategy allows for selective oncolysis of tumors cells and the transgenes mediate potent stimulation of systemic anti-tumor immunity. Since human IL-12 and the anti-CTLA-4 antagonist do not cross-react with the mouse targets, most nonclinical pharmacology studies were conducted with the mouse surrogate mONCR-171, which expresses within the same base vector as ONCR-177 the mouse equivalent to the ONCR-177 transgenes. Intra-tumoral administration of mONCR-171 in the oHSV-sensitive A20 BALB/c lymphoma bilateral tumor model resulted in response rates (partial and complete tumor regressions) of 100% and 80%, respectively, on the injected (ipsilateral) and distant (contralateral) tumor. mONCR-171 was also highly efficacious in the B16F10 melanoma model, an oHSV-resistant C57BL/6 based tumor model engineered to be permissive to oHSV by introduction of Nectin-1, and in the oHSV-resistant MC38 C57BL/6 colon carcinoma model. Abscopal anti-tumor activity could not be explained by propagation of the virus since viral DNA and transgenes were only detectable in the injected tumor. Rather, mONCR-171 treatment resulted in increased numbers of activated NK cells, CD8 and CD4 T cells, and classical dendritic cells. The proportion of Tregs decreased, resulting in large CD8/Treg ratios. These changes in immune contexture occurred in both the ipsilateral and contralateral tumor and were more pronounced with mONCR-171 treatment compared to the base vector without transgenes, indicating that the observed abscopal effects were due to the elicitation of systemic anti-tumor immunity mediated in part by the transgenes. These encouraging preclinical data warrant the clinical investigation of ONCR-177 in patients with metastatic cancer. Citation Format: Brian B. Haines, Agnieszka Denslow, Michael S. Ball, Jacqueline Gursha, Daniel Wambua, Cecilia Kwong, Lingxin Kong, Prajna Behera, Peter Grzesik, Caitlin Goshert, Allison Colthart, Jennifer S. Lee, Terry Farkaly, Edward M. Kennedy, Lorena Lerner, Christophe Queva. Development of ONCR-177, an miR-attenuated oncolytic HSV-1 designed to potently activate systemic antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 940.

Isolation of a naturally occurring vaccine/wild-type recombinant bovine herpesvirus type 1 (BoHV-1) from an aborted bovine fetus

Bovine herpesvirus type 1 (BoHV-1) causes various disease syndromes in cattle including respiratory disease and abortions. During an investigation into the potential role of BoHV-1 modified-live vaccines (MLV) causing diseases in cattle, we performed whole genome sequencing on six BoHV-1 field strains isolated at Cornell Animal Health Diagnostic Center in the late 1970s. Three isolates (two respiratory and a fetal) were identified as vaccine-derived isolates, having SNP patterns identical to that of a previously sequenced MLV virus that exhibited a deleted US2 and truncated US1.67 genes. Two other isolates (a respiratory and a fetal) were categorized as wild-type (WT) viruses based on their unique SNP pattern that is distinct from MLV viruses. The sixth isolate from an aborted fetus was a recombinant virus with 62% of its genome exhibiting SNPs identical to one of the above-mentioned WT viruses also recovered from an aborted fetus. The remaining 38% consisted of two blocks of sequences derived from the MLV virus. The first block replaced the UL9-UL19 region, and the second vaccine-derived sequence block encompassed all the genes within the unique short region and the internal/terminal repeats containing the regulatory genes BICP4 and BICP22. This is confirmatory evidence that recombination between BoHV-1 MLV and WT viruses can occur under natural conditions and cause disease. It is important in that it underscores the potential for the glycoprotein E negative (gE−) marker vaccine used to eradicate BoHV-1 in some countries, to recombine with virulent field strains allowing them to capture the gE− marker, thereby endangering the control and eradication programs.

Immune responses upon in ovo HVT-IBD vaccination vary between different chicken lines

The genotype of chickens is assumed to be associated with variable immune responses. In this study a modern, moderate performing dual-purpose chicken line (DT) was compared with a high-performing layer-type (LT) as well as a broiler-type (BT) chicken line. One group of each genotype was vaccinated in ovo with a recombinant herpesvirus of turkeys expressing the virus protein VP2 of the infectious bursal disease virus (HVT-IBD) while one group of each genotype was left HVT-IBD unvaccinated (control group). Genotype associated differences in innate and adapted immune responses between the groups were determined over five weeks post hatch. HVT-IBD vaccination significantly enhanced humoral immune responses against subsequently applied live vaccines compared to non-HVT-IBD vaccinated groups at some of the investigated time points (P < 0.05). In addition HVT-IBD vaccination had depending on the genotype a significant impact on splenic macrophage as well as bursal CD4+ T-cell numbers (P < 0.05). On the other hand, the detectable genotype influence on Interferon (IFN) γ and nitric oxide (NO) release of ex vivo stimulated spleen cells was independent of HVT-IBD vaccination. The results of our study suggest considering a genotype specific vaccination regime in the field.

A recombinant bovine herpesvirus-4 vectored vaccine delivered via intranasal nebulization elicits viral neutralizing antibody titers in cattle.

Recombinant herpesvirus vaccine vectors offer distinct advantages in next-generation vaccine development, primarily due to the ability to establish persistent infections to provide sustainable antigen responses in the host. Recombinant bovine herpesvirus-4 (BoHV-4) has been previously shown to elicit protective immunity in model laboratory animal species against a variety of pathogens. For the first time, we describe the induction of antigen-specific immune responses to two delivered antigens in the host species after intranasal nebulization of recombinant BoHV-4 expressing the chimeric peptide containing the bovine viral diarrhea virus (BVDV) glycoprotein E2 and the bovine herpesvirus 1 (BoHV-1) glycoprotein D (BoHV-4-A-CMV-IgK-gE2gD-TM). In this study, four cattle were immunized via intranasal nebulization with the recombinant BoHV-4 construct. Two of the cattle were previously infected with wild-type BoHV-4, and both developed detectable serologic responses to BVDV and BoHV-1. All four immunized cattle developed detectable viral neutralizing antibody responses to BVDV, and one steer developed a transient viral neutralizing response to BoHV-1. Approximately one year after immunization, immunosuppressive doses of the glucocorticoid dexamethasone were administered intravenously to all four cattle. Within two weeks of immunosuppression, all animals developed viral neutralizing antibody responses to BoHV-1, and all animals maintained BVDV viral neutralizing capacity. Overall, nebulization of BoHV-4-A-CMV-IgK-gE2gD-TM persistently infects cattle, is capable of eliciting antigen-specific immunity following immunization, including in the presence of pre-existing BoHV-4 immunity, and recrudescence of the virus boosts the immune response to BoHV-4-vectored antigens. These results indicate that BoHV-4 is a viable and attractive vaccine delivery platform for use in cattle.

Protection Efficacy of a Recombinant Herpesvirus of Turkey Vaccine Against Infectious Laryngotracheitis Virus Administered In Ovo to Broilers at Three Standardized Doses.

Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens that produces significant economic losses to the poultry industry. The disease is caused by Gallid alpha herpesvirus-1 (GaHV-1), commonly known as the infectious laryngotracheitis virus (ILTV). Vaccination remains necessary for the control of the disease. Due to the inherent virulence of live attenuated vaccines, in particular that of the chicken embryo origin (CEO) vaccines, the use of ILT viral vector recombinant vaccines has significantly expanded worldwide as a safer vaccination strategy. However, the protective efficacy of recombinant ILT vaccines can be compromised by the use of fractional doses and improper handling and administration of the vaccine. The objective of this study was twofold: 1) to evaluate the protection efficacy induced by a commercial recombinant HVT-LT (rHVT-LT) vaccine when administered in ovo to broilers at three standardized doses (6000 plaque-forming units [PFU], 3000 PFU, and 1000 PFU), and 2) to assess the potential of rHVT-LT-vaccinated chickens to spread virus to contact chickens after challenge. Independently of the vaccine dose, vaccinated chickens showed reduction in clinical signs, maintained body weight gain after challenge, and lessened the challenge virus replication in the trachea at a rate of 52%-65%. However, in spite of this reduction, transmission of challenge virus from rHVT-LT-vaccinated (6000/Ch, 3000/Ch) to contact-naive chickens was evident. This study is the first to support that rHVT-LT vaccination did not prevent spread of challenge virus to contact birds.

A double recombinant herpes virus of turkeys for the protection of chickens against Newcastle, infectious laryngotracheitis and Marek’s diseases

ABSTRACTA double recombinant strain of herpes virus of turkeys (HVT) was constructed that contains the fusion (F) gene from Newcastle disease virus (NDV) and the gD plus gI genes from infectious laryngotracheitis virus (ILTV) inserted into a non-essential region of the HVT genome. Expression of the F protein was controlled by a human cytomegalovirus promoter, whereas expression of gD plus gI was driven by an ILTV promoter. The double recombinant vaccine virus (HVT-NDV-ILT) was fully stable genetically and phenotypically following extended passage in cell culture and infection of chickens. Safety of the vaccine virus was confirmed by overdose and backpassage studies in specific-pathogen-free chickens. Chickens vaccinated with a single dose of HVT-NDV-ILT administered by the in ovo route were highly protected from challenge with the velogenic NDV (GB Texas), ILTV (LT 96-3) and Marek’s disease virus (GA 5) strains (97%, 94% and 97%, respectively). Similarly, chickens vaccinated with a single dose by subcutaneous (SC) route at 1 day of age were highly protected from challenge with the same three viruses (100%, 100%, and 88%, respectively). The protection level of a single dose given by in ovo or SC route against challenge with a virulent Marek’s disease virus strain demonstrates that insertion of multiple genes from two different pathogens within the HVT genome had no adverse effect on the capacity of HVT to protect against Marek’s disease. These results demonstrate that HVT-NDV-ILT is a safe and efficacious vaccine for simultaneous control of NDV, ILTV and Marek’s diseases.

Efficacy of a Recombinant Turkey Herpesvirus AI (H5) Vaccine in Preventing Transmission of Heterologous Highly Pathogenic H5N8 Clade 2.3.4.4b Challenge Virus in Commercial Broilers and Layer Pullets.

Outbreaks caused by the highly pathogenic avian influenza virus (HPAIV) H5N8 subtype clade 2.3.4.4 were first reported in 2014 in South Korea then spread very rapidly in Asia, to Europe, and for the first time, to North America. Efficacy of a recombinant HVT-AI (H5) vaccine (rHVT-H5) to provide clinical protection as well as to significantly reduce the shedding of an H5N8 challenge virus has already been demonstrated in SPF chickens. The aim of our studies was to test the efficacy of the same rHVT-H5 vaccine in controlling the transmission of a recent Hungarian HPAIV H5N8 challenge virus in commercial chickens. Broilers and layers were vaccinated at day old according to the manufacturer's recommendation and then challenged with a 2017 Hungarian HPAIV H5N8 (2.3.4.4b) isolate at 5 or 7 weeks of age, respectively. Evaluation of clinical protection, reduction of challenge virus shedding, and transmission to vaccinated contact birds was done on the basis of clinical signs/mortality, detection, and quantitation of challenge virus in oronasal and cloacal swabs (regularly between 1 and 14 days postchallenge). Measurement of seroconversion to AIV nucleoprotein was used as an indicator of infection and replication of challenge virus. Our results demonstrated that rHVT-H5 vaccination could prevent the development of clinical disease and suppress shedding very efficiently, resulting in the lack of challenge virus transmission to vaccinated contact chickens, regardless the type of birds. Single immunization with the tested rHVT-H5 vaccine proved to be effective to stop HPAIV H5N8 (2.3.4.4b) transmission within vaccinated poultry population under experimental conditions.

Maternal antibody inhibition of recombinant Newcastle disease virus vectored vaccine in a primary or booster avian influenza vaccination program of broiler chickens

Maternally-derived antibodies (MDA) provide early protection from disease, but may interfere with active immunity in young chicks. In highly pathogenic avian influenza virus (HPAIV)-enzootic countries, broiler chickens typically have MDA to Newcastle disease virus (NDV) and H5 HPAIV, and their impact on active immunity from recombinant vectored vaccines is unclear. We assessed the effectiveness of a spray-applied recombinant NDV vaccine with H5 AIV insert (rNDV-H5) and a recombinant turkey herpesvirus (HVT) vaccine with H5 AIV insert (rHVT-H5) in commercial broilers with MDA to NDV alone (MDA:AIV−NDV+) or to NDV plus AIV (MDA:AIV+NDV+) to provide protection against homologous HPAIV challenge. In Experiment 1, chicks were spray-vaccinated with rNDV-H5 at 3 weeks (3w) and challenged at 5 weeks (5w). All sham-vaccinated progeny lacked AIV antibodies and died following challenge. In rNDV-H5 vaccine groups, AIV and NDV MDA had completely declined to non-detectable levels by vaccination, enabling rNDV-H5 spray vaccine to elicit a protective AIV antibody response by 5w, with 70–78% survival and significant reduction of virus shedding compared to shams. In Experiment 2, progeny were vaccinated with rHVT-H5 and rNDV-H5 at 1 day (1d) or 3w and challenged at 5w. All sham-vaccinated progeny lacked AIV antibodies and died following challenge. In rHVT-H5(1d) vaccine groups, irrespective of rNDV-H5(3w) boost, AIV antibodies reached protective levels pre-challenge, as all progeny survived and virus shedding significantly decreased compared to shams. In contrast, rNDV-H5-vaccinated progeny had AIV and/or NDV MDA at the time of vaccination (1d and/or 3w) and failed to develop a protective immune response by 5w, resulting in 100% mortality after challenge. Our results demonstrate that MDA to AIV had minimal impact on the effectiveness of rHVT-H5, but MDA to AIV and/or NDV at the time of vaccination can prevent development of protective immunity from a primary or booster rNDV-H5 vaccine.

N-terminal domain of Schmallenberg virus envelope protein Gc delivered by recombinant equine herpesvirus type 1 and modified vaccinia virus Ankara: Immunogenicity and protective efficacy in cattle

Schmallenberg virus (SBV), which emerged in 2011 in Central Europe and subsequently spread very rapidly throughout the continent, affects predominantly ruminants. SBV is transmitted by insect vectors, and therefore vaccination is one of the major tools of disease control. Only recently, a domain connected to virus neutralization has been identified at the amino-terminal part of the viral envelope protein Gc. Here, this Gc domain delivered by recombinant EHV-1 or MVA vector viruses was tested in a vaccination-challenge trial in cattle, one of the major target species of SBV.The EHV-1-based vaccine conferred protection in two of four animals, whereas immunization using the MVA vector vaccine efficiently induced an SBV-specific antibody response and full protection against SBV challenge infection in all the vaccinated animals. Moreover, due to the absence of antibodies against SBVs N-protein, both vector vaccines enable the differentiation between vaccinated and field-infected animals making them to a promising tool to control SBV spread as well as to prevent disease in domestic ruminants.

Intrauterine vaccination induces a dose-sensitive primary humoral response with limited evidence of recall potential.

Induction of the local mucosal immune system within the reproductive tract is widely considered to be a key component in the development of effective prophylactic vaccines to control the spread of sexually transmitted infections. Here, we examine the capacity of the upper reproductive tract to act as a site of immune induction following. Two vaccines formulated with a triple adjuvant combination and either recombinant bovine herpesvirus (tgD) protein or ovalbumin (OVA) were delivered at varying doses to the uterine lumen of rabbits and the resulting immune response evaluated after 32days. Intrauterine vaccination produced a dose-dependent induction of both antigen-specific IgG and IgA in serum. Both uterine and broncheoalveolar lavage of the high and medium-dose vaccine group contained a significant increase in both anti-OVA and anti-tgD IgG, but no significant quantities of antigen-specific IgA were observed. The restimulation of splenocytes from the high-dose vaccine group with ovalbumin (OVA) only resulted in a small but significant increase in gene expression of the Th1 cytokines (IL2/IFNγ) in the absence of an observable increase in proliferation. Collectively, the results confirm the capacity of the uterine immune system to generate a primary response following stimulation.

Early immune responses and profiling of cell-mediated immunity-associated gene expression in response to rHVT-IBD vaccination

Infectious bursal disease (IBD) remains a major threat to the poultry industry. Recombinant herpesvirus of turkey (rHVT)-IBD vaccines have been successfully used to induce a protective immune response against IBD. However, the capacity for rHVT-IBD vaccines to induce early protection without detectable antibodies, and the underlying mechanisms mediating specific cell-mediated responses in the early stages following vaccination, have been poorly investigated. Therefore, in this study, specific pathogen-free (SPF) chickens were vaccinated with rHVT-IBD and T-cell subsets were analyzed. Both splenic and circulating CD8+ cell populations increased at 7 days postvaccination (dpv). Next, the expression of adaptive immunity-related genes was analyzed in the spleen and lung of rHVT-IBD-vaccinated chickens. Upregulation of CD8 expression was observed at 7 dpv. Interestingly, a parallel increase in the transcription of granzymes A and K was also detected from 7 dpv. To our knowledge, the latter result is the first to be reported, and it suggests that cytotoxic activity of CD8+ T lymphocytes is activated. In contrast, expression of the innate genes examined remained largely unchanged following vaccination. To further investigate the IBD virus (IBDV)-specific responses triggered by rHVT-IBD vaccination, vaccinated chickens were inoculated with an attenuated IBDV strain with the aim of restimulating induced immune responses in vivo. The expression profiles of various genes associated with adaptive immune responses were subsequently analyzed in lung, spleen, and bursa of Fabricius samples. Significant upregulation of CD4, CD8, perforin, and IFNγ expression were observed in the bursa samples 7 days postinoculation (dpi). In the lung, transcript levels of CD8, granzymes and perforin were also significantly higher in the rHVT-IBD-vaccinated chickens at 7 dpi, thereby suggesting that specific cellular immune responses were activated. Overall, these results support the hypothesis that stimulation of specific CD8+ cell-mediated immunity contributes to the response against IBDV in rHVT-IBD-vaccinated chickens.

A simple and rapid approach to develop recombinant avian herpesvirus vectored vaccines using CRISPR/Cas9 system

Herpesvirus of turkeys (HVT) has been successfully used as live vaccine against Marek's disease (MD) worldwide for more than 40 years either alone or in combination with other serotypes. HVT is also widely used as a vector platform for generation of recombinant vaccines against a number of avian diseases such as infectious bursal disease (IBD), Newcastle disease (ND) and avian influenza (AI) using conventional recombination methods or recombineering tools on cloned viral genomes. In the present study, we describe the application of CRISPR/Cas9-based genome editing as a rapid and efficient method of generating HVT recombinants expressing VP2 protein of IBDV. This approach offers an efficient method to introduce other viral antigens into the HVT genome for rapid development of recombinant vaccines.

A Promising Recombinant Herpesvirus of Turkeys Vaccine Expressing PmpD-N of Chlamydia psittaci Based on Elongation Factor-1 Alpha Promoter.

The obligate intracellular Gram-negative bacterium Chlamydia psittaci often causes avian chlamydiosis and influenza-like symptoms in humans. However, the commercial subunit C. psittaci vaccine could only provide a partial protection against avian chlamydiosis due to poor cellular immune response. In our previous study, a recombinant herpesvirus of turkeys (HVT)-delivered vaccine against C. psittaci and Marek’s disease based on human cytomegalovirus (CMV) promoter (rHVT-CMV-pmpD) was developed and provided an effective protection against C. psittaci disease with less lesions and reduced chlamydial loads. In this study, we developed another recombinant HVT vaccine expressing the N-terminal fragment of PmpD (PmpD-N) based on human elongation factor-1 alpha (EF-1α) promoter (rHVT-EF-pmpD) by modifying the HVT genome within a bacterial artificial chromosome. The related characterization of rHVT-EF-pmpD was evaluated in vitro in comparison with that of rHVT-CMV-pmpD. The expression of PmpD-N was determined by western blot. Under immunofluorescence microscopy, PmpD-N protein of both two recombinant viruses was located in the cytoplasm and on the cell surface. Growth kinetics of rHVT-EF-pmpD was comparable to that of rHVT-CMV-pmpD, and the growth rate of rHVT-EF-pmpD was apparently higher than that of rHVT-CMV-pmpD on 48, 72, and 120 h postinfection. Macrophages activated by rHVT-EF-pmpD could produce more nitric oxide and IL-6 than that activated by rHVT-CMV-pmpD. In this study, a recombinant HVT vaccine expressing PmpD-N based on EF-1α promoter was constructed successfully, and a further research in vivo was needed to analyze the vaccine efficacy.

The efficacy of recombinant turkey herpesvirus vaccines targeting the H5 of highly pathogenic avian influenza virus from the 2014–2015 North American outbreak

The outbreak of highly pathogenic avian influenza virus in North American poultry during 2014 and 2015 demonstrated the devastating effects of the disease and highlighted the need for effective emergency vaccine prevention and control strategies targeted at currently circulating strains. This study evaluated the efficacy of experimental recombinant turkey herpesvirus vector vaccines with three different inserts targeting the hemagglutinin gene of an isolate from the recent North American influenza outbreak. White leghorn chickens were vaccinated at one day of age and challenged with A/Turkey/Minnesota/12582/2015 H5N2 at 4 weeks of age. Birds were analyzed for survival, viral shedding at two and four days after infection, and specific antibody prior to challenge and from surviving birds. The three experimental vaccines demonstrated 100%, 45% and 15% survival with the most effective vaccine significantly reducing oral and cloacal viral shedding compared to all other groups and generated specific antibody prior to challenge with highly pathogenic avian influenza virus. More studies are needed using diverse H5Nx highly pathogenic virus isolates to fully determine the breadth of coverage against possible exposure strains, as well as possible impact of maternally derived antibody on protection and vaccine efficacy.

Protection of commercial turkeys following inactivated or recombinant H5 vaccine application against the 2015U.S. H5N2 clade 2.3.4.4 highly pathogenic avian influenza virus.

Between December 2014 and June 2015, North America experienced the largest recorded foreign animal disease outbreak with over 47 million poultry dead or euthanized from viral exposure to a clade 2.3.4.4 H5 highly pathogenic avian influenza (HPAI) epizootic. Soon after the epizootic began, the U.S. Department of Agriculture (USDA) began testing the efficacy of different vaccines as a possible future control strategy. The aim of these studies were to evaluate the efficacy three H5 vaccines to aid in control of HPAI in commercial turkeys. Three different vaccine technologies were evaluated for efficacy: 1) inactivated reverse genetic laboratory-generated virus encoding a clade 2.3.4.4 H5 hemagglutinin (HA) gene (rgH5), 2) recombinant turkey herpesvirus encoding a clade 2.2. H5 HA (rHVT-AI), and 3) recombinant replication-deficient alphavirus RNA particle vaccine encoding a clade 2.3.4.4 H5 HA (RP-H5). All vaccines tested significantly (P<0.01) increased survival rates between vaccinated and sham vaccinated groups of poults challenged with A/turkey/Minnesota/12582/2015 clade 2.3.4.4 H5N2 HPAI. The rgH5 vaccine had detectable serum hemagglutination inhibition (HI) antibody against the challenge virus, and significantly reduced the frequency and level of viral shedding from oropharyngeal and cloacal swabs at days 2 and 4 post-challenge. Vaccination with only rHVT-AI or RP-H5 was not 100% protective, and failed to significantly reduce viral shedding post-challenge. A combined prime and boost strategy with the rHVT-AI and RP-H5, or rHVT-AI and rgH5, was 100% protective against lethal H5N2 HPAI challenge. Results of these studies led to USDA conditional approval of commercially available recombinant vaccines for use in turkeys as a control measure for clade 2.3.4.4 H5 HPAI epizootics.

Use of a Recombinant Gamma-2 Herpesvirus Vaccine Vector against Dengue Virus in Rhesus Monkeys.

Research on vaccine approaches that can provide long-term protection against dengue virus infection is needed. Here we describe the construction, immunogenicity, and preliminary information on the protective capacity of recombinant, replication-competent rhesus monkey rhadinovirus (RRV), a persisting herpesvirus. One RRV construct expressed nonstructural protein 5 (NS5), while a second recombinant expressed a soluble variant of the E protein (E85) of dengue virus 2 (DENV2). Four rhesus macaques received a single vaccination with a mixture of both recombinant RRVs and were subsequently challenged 19 weeks later with 1 × 105 PFU of DENV2. During the vaccine phase, plasma of all vaccinated monkeys showed neutralizing activity against DENV2. Cellular immune responses against NS5 were also elicited, as evidenced by major histocompatibility complex class I (MHC-I) tetramer staining in the one vaccinated monkey that was Mamu-A*01 positive. Unlike two of two unvaccinated controls, two of the four vaccinated monkeys showed no detectable viral RNA sequences in plasma after challenge. One of these two monkeys also showed no anamnestic increases in antibody levels following challenge and thus appeared to be protected against the acquisition of DENV2 following high-dose challenge. Continued study will be needed to evaluate the performance of herpesviral and other persisting vectors for achieving long-term protection against dengue virus infection.IMPORTANCE Continuing studies of vaccine approaches against dengue virus (DENV) infection are warranted, particularly ones that may provide long-term immunity against all four serotypes. Here we investigated whether recombinant rhesus monkey rhadinovirus (RRV) could be used as a vaccine against DENV2 infection in rhesus monkeys. Upon vaccination, all animals generated antibodies capable of neutralizing DENV2. Two of four vaccinated monkeys showed no detectable viral RNA after subsequent high-dose DENV2 challenge at 19 weeks postvaccination. Furthermore, one of these vaccinated monkeys appeared to be protected against the acquisition of DENV2 infection on the basis of undetectable viral loads and the lack of an anamnestic antibody response. These findings underscore the potential utility of recombinant herpesviruses as vaccine vectors.

Recombinant Gallid herpesvirus 2 with interrupted meq genes confers safe and efficacious protection against virulent field strains.

Gallid herpesvirus 2 (GaHV-2) continuously evolves, which reduces the effectiveness of existing vaccines. To construct new GaHV-2 candidate vaccines, LMS, which is a virulent GaHV-2 field strain isolated from diseased chicken flocks in Southwest China in 2007, was modified such that both copies of its meq oncogene were partially deleted. The resulting virus, i.e., rMSΔmeq, was characterized using PCR and sequencing. To evaluate the safety and protective efficacy of rMSΔmeq, specific pathogen-free (SPF) chickens were inoculated with 2000 plaque forming units (pfu) and 20,000pfu of rMSΔmeq immediately after hatching. All birds grew well during the experimental period, and none of the challenged chickens developed Marek's disease-associated lymphoma. In addition, the rMSΔmeq- and CVI988/Rispens-vaccinated SPF chickens were challenged with 1000 pfu and 5000 pfu of the representative virulent GaHV-2 Md5 strain and 1000 pfu of the variant GaHV-2 strains LCC or LTS. The results showed that the rMSΔmeq strain provided complete protection, which was similar to that provided by the CVI988/Rispens vaccine (protective index (PI) of 95.5) when challenged with a conventional dose of the Md5 strain. However, rMSΔmeq provided a PI of 90.9 when challenged with 5000 pfu of the Md5 strain, which was significantly higher than that provided by the CVI988/Rispens vaccine (54.5). rMSΔmeq provided a PI of 86.4 against LCC, which was equal to that provided by the CVI988/Rispens vaccine (81.8). In addition, rMSΔmeq provided a PI of 100 against LTS, which was significantly higher than that provided by the CVI988/Rispens vaccine (68.2). Altogether, the rMSΔmeq virus provided efficient protection against representative and variant GaHV-2 strains. In conclusion, the rMSΔmeq virus is a safe and effective vaccine candidate for the prevention of Marek's disease and is effective against the Chinese variant GaHV-2 strains.