Live Vaccine Vector Research Articles

A Quadruple Gene-Deleted Live BoHV-1 Subunit RVFV Vaccine Vector Reactivates from Latency and Replicates in the TG Neurons of Calves but Is Not Transported to and Shed from Nasal Mucosa.

Bovine herpesvirus type 1 (BoHV-1) establishes lifelong latency in trigeminal ganglionic (TG) neurons following intranasal and ocular infection in cattle. Periodically, the latent virus reactivates in the TG due to stress and is transported anterogradely to nerve endings in the nasal epithelium, where the virus replicates and sheds. Consequently, BoHV-1 is transmitted to susceptible animals and maintained in the cattle population. Modified live BoHV-1 vaccine strains (BoHV-1 MLV) also have a similar latency reactivation. Therefore, they circulate and are maintained in cattle herds. Additionally, they can regain virulence and cause vaccine outbreaks because they mutate and recombine with other circulating field wild-type (wt) strains. Recently, we constructed a BoHV-1 quadruple mutant virus (BoHV-1qmv) that lacks immune evasive properties due to UL49.5 and glycoprotein G (gG) deletions. In addition, it also lacks the gE cytoplasmic tail (gE CT) and Us9 gene sequences designed to make it safe, increase its vaccine efficacy against BoHV-1, and restrict its anterograde neuronal transport noted above. Further, we engineered the BoHV-1qmv-vector to serve as a subunit vaccine against the Rift Valley fever virus (BoHV-1qmv Sub-RVFV) (doi: 10.3390/v15112183). In this study, we determined the latency reactivation and nasal virus shedding properties of BoHV-1qmv (vector) and BoHV-1qmv-vectored subunit RVFV (BoHV-1qmv sub-RVFV) vaccine virus in calves in comparison to the BoHV-1 wild-type (wt) following intranasal inoculation. The real-time PCR results showed that BoHV-1 wt- but not the BoHV-1qmv vector- and BoHV-1qmv Sub-RVFV-inoculated calves shed virus in the nose following dexamethasone-induced latency reactivation; however, like the BoHV-1 wt, both the BoHV-1qmv vector and BoHV-1qmv Sub-RVFV viruses established latency, were reactivated, and replicated in the TG neurons. These results are consistent with the anterograde neurotransport function of the gE CT and Us9 sequences, which are deleted in the BoHV-1qmv and BoHV-1qmv Sub-RVFV.

Recent Advances in the Study of Alphaherpesvirus Latency and Reactivation: Novel Guidance for the Design of Herpesvirus Live Vector Vaccines.

Alphaherpesviruses, including herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV), infect a diverse array of hosts, spanning both humans and animals. Alphaherpesviruses have developed a well-adapted relationship with their hosts through long-term evolution. Some alphaherpesviruses exhibit a typical neurotropic characteristic, which has garnered widespread attention and in-depth research. Virus latency involves the retention of viral genomes without producing infectious viruses. However, under stress, this can be reversed, resulting in lytic infection. Such reactivation events can lead to recurrent infections, manifesting as diseases like herpes labialis, genital herpes, and herpes zoster. Reactivation is a complex process influenced by both viral and host factors, and identifying how latency and reactivation work is vital to developing new antiviral therapies. Recent research highlights a complex interaction among the virus, neurons, and the immune system in regulating alphaherpesvirus latency and reactivation. Neurotropic alphaherpesviruses can breach host barriers to infect neurons, proliferate extensively within their cell bodies, and establish latent infections or spread further. Whether infecting neurons or spreading further, the virus undergoes transmission along axons or dendrites, making this process an indispensable part of the viral life cycle and a critical factor influencing the virus's invasion of the nervous system. Research on the transmission process of neurotropic alphaherpesviruses within neurons can not only deepen our understanding of the virus but can also facilitate the targeted development of corresponding vaccines. This review concentrates on the relationship between the transmission, latency, and activation of alphaherpesviruses within neurons, summarizes recent advancements in the field, and discusses how these findings can inform the design of live virus vaccines for alphaherpesviruses.

Research Progress in the Development of Vaccines against Mycoplasma gallisepticum and Mycoplasma synoviae.

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the primary agents responsible for mycoplasma disease in poultry. MG has been identified as a significant cause of chronic respiratory disease in chickens, while MS has been linked to the development of tenosynovitis, joint swelling and other symptoms in chickens, leading to considerable economic losses for the poultry industry. Unfortunately, there is no specific drug for treatment and vaccination is the most important way to control the disease. There are some different types of vaccines, including live vaccines, inactivated vaccines, sub-unit vaccines and vector vaccines. This paper provides a comprehensive review of the development of vaccines for MG and MS.

Genetic engineering of Salmonella spp. for novel vaccine strategies and therapeutics.

Salmonella enterica is a diverse species that infects both humans and animals. S. enterica subspecies enterica consists of more than 1,500 serovars. Unlike typhoidal Salmonella serovars which are human host-restricted, non-typhoidal Salmonella (NTS) serovars are associated with foodborne illnesses worldwide and are transmitted via the food chain. Additionally, NTS serovars can cause disease in livestock animals causing significant economic losses. Salmonella is a well-studied model organism that is easy to manipulate and evaluate in animal models of infection. Advances in genetic engineering approaches in recent years have led to the development of Salmonella vaccines for both humans and animals. In this review, we focus on current progress of recombinant live-attenuated Salmonella vaccines, their use as a source of antigens for parenteral vaccines, their use as live-vector vaccines to deliver foreign antigens, and their use as therapeutic cancer vaccines in humans. We also describe development of live-attenuated Salmonella vaccines and live-vector vaccines for use in animals.

The JMU-SalVac-System: A Novel, Versatile Approach to Oral Live Vaccine Development.

Salmonella enterica Serovar Typhi Ty21a (Ty21a) is the only licensed oral vaccine against typhoid fever. Due to its excellent safety profile, it has been used as a promising vector strain for the expression of heterologous antigens for mucosal immunization. As the efficacy of any bacterial live vector vaccine correlates with its ability to express and present sufficient antigen, the genes for antigen expression are traditionally located on plasmids with antibiotic resistance genes for stabilization. However, for use in humans, antibiotic selection of plasmids is not applicable, leading to segregational loss of the antigen-producing plasmid. Therefore, we developed an oral Ty21a-based vaccine platform technology, the JMU-SalVac-system (Julius-Maximilians-Universität Würzburg) in which the antigen delivery plasmids (pSalVac-plasmid-series) are stabilized by a ΔtyrS/tyrS+-based balanced-lethal system (BLS). The system is made up of the chromosomal knockout of the essential tyrosyl-tRNA-synthetase gene (tyrS) and the in trans complementation of tyrS on the pSalVac-plasmid. Further novel functional features of the pSalVac-plasmids are the presence of two different expression cassettes for the expression of protein antigens. In this study, we present the construction of vaccine strains with BLS plasmids for antigen expression. The expression of cytosolic and secreted mRFP and cholera toxin subunit B (CTB) proteins as model antigens is used to demonstrate the versatility of the approach. As proof of concept, we show the induction of previously described in vivo inducible promoters cloned into pSalVac-plasmids during infection of primary macrophages and demonstrate the expression of model vaccine antigens in these relevant human target cells. Therefore, antigen delivery strains developed with the JMU-SalVac technology are promising, safe and stable vaccine strains to be used against mucosal infections in humans.

Immunoadjuvant Effect of Chitosan Oligosaccharide and Its Feasibility of Being Used as an Adjuvant for Attenuated Live Bacteria Vector Vaccines

To study the immunoadjuvant effects of chitosan oligosaccharide (COS), including the immune activation and the triggering of lysosomal escape, and to explore whether COS can be used as an adjuvant for attenuated live bacteria vector vaccines. 1) Mouse macrophages RAW264.7 cells were cultured with COS at 0 mg/mL (the control group) and 0.1-4 mg/mL for 24 h and the effect on cell viability was measured by CCK8 assay. Mouse macrophages RAW264.7 were treated with COS at 0 (the control group), 1, 2, and 4 mg/mL for 24 h. Then, the mRNA expression levels of the cytokines, including IFN-γ, IL-10, TGF-β, and TLR4, were determined by RT-qPCR assay. 2) RAW264.7 cells were treated with 1 mL of PBS containing different components, including calcein at 50 μg/mL, COS at 2 mg/mL, and bafilomycin A1, an inhibitor, at 1 μmol/mL, for culturing. The cells were divided into the Calcein group, Calcein+COS group, and Calcein+COS+Bafilomycin A1 group accordingly. Laser scanning confocal microscopy was used to observe the phagocytosis and the intracellular fluorescence distribution of calcein, a fluorescent dye, in RAW264.7 cells in the presence or absence of COS intervention to determine whether COS was able to trigger lysosomal escape. 3) LM∆E6E7 and LI∆E6E7, the attenuated Listeria vector candidate therapeutic vaccines for cervical cancer, were encapsulated with COS at the mass concentrations of 0.5 mg/mL, 1 mg/mL, 2 mg/mL , 4 mg/mL, and 8 mg/mL. Then, the changes in zeta potential were measured to select the concentration of COS that successfully encapsulated the bacteria. Phagocytosis of the vaccine strains by RAW264.7 cells was measured before and after LM∆E6E7 and LI∆E6E7 were coated with COS at 2 mg/mL. 1) CCK8 assays showed that, compared with the findings for the control group, the intervention of RAW264.7 cells with COS at different concentrations for 24 h was not toxic to the cells and promoted cell proliferation, with the difference being statistically significant (P<0.05). According to the RT-qPCR results, compared with those of the control group, the COS intervention up-regulated the mRNA levels of TLR4 and IFN-γ in RAW264.7 cells, while it inhibited the mRNA expression levels of TGF-β and IL-10, with the most prominent effect being observed in the 4 mg/mL COS group (P<0.05). 2) Laser scanning confocal microscopy revealed that the amount of fluorescent dye released from lysosomes into the cells was greater in the Calcein+COS group than that in the Calcein group. In other words, a greater amount of fluorescent dye was released from lysosomes into the cells under COS intervention. Furthermore, this process could be blocked by bafilomycin A1. 3) The zeta potential results showed that COS could successfully encapsulate the surface of bacteria when its mass concentration reached 2 mg/mL. Before and after the vaccine strain was encapsulated by COS, the phagocytosis of LM∆E6E7 by RAW264.7 cells was 5.70% and 22.00%, respectively, showing statistically significant differences (P<0.05); the phagocytosis of LI∆E6E7 by RAW264.7 cells was 1.55% and 6.12%, respectively, showing statistically significant differences (P<0.05). COS has the effect of activating the immune response of macrophages and triggering lysosomal escape. The candidates strains of coated live attenuated bacterial vector vaccines can promote the phagocytosis of bacteria by macrophages. Further research is warranted to develop COS into an adjuvant for bacterial vector vaccine.

The booster immunization using commercial vaccines effectively protect chickens against novel variants of infectious bursal disease virus (genotype A2dB1)

The novel variant IBDV (nVarIBDV, genotype A2dB1), characterized by bursal atrophy of fabricius and decreased lymphocytes, has been emerging on a large scale in Asia (including China) since late 2018. nVarIBDV is a new threat to the poultry industry, yet the currently licensed commercial vaccines, including the live viral vector vaccine, IBDV immune complex vaccine or VP2 subunit vaccine, are ineffective against nVarIBDV infection. In this study, specific-pathogen-free (SPF) chickens and broilers divided into three groups were vaccinated with the live viral vector vaccine, the VP2 subunit vaccine or the IBDV immune complex vaccine at 1 day old, respectively. The SPF chickens received a secondary vaccination with the live B87 strain vaccine at 11 days-old. The bursa/body weight ratio, histopathology lesion of the bursa, and the differentiation between infected and vaccinated animals (DIVA) by qRT-PCR confirmed that the live viral vector vaccine or immune complex vaccine plus live B87 strain booster could provide at least 80% protection against the FJ2019-01 strain of nVarIBDV in SPF chickens. The broilers also received a secondary vaccination using a live W2512 G-61 strain vaccine at 14 days-old, and analyses showed that the VP2 subunit vaccine or immune complex vaccine plus the live W2512 G-61 strain booster also provided more than 80% protection against the FJ2019-01 strain of nVarIBDV. Unfortunately, the live viral vector vaccine plus live W2512 G-61 strain booster provided poor to moderate protection against FJ2019-01 in broilers. These findings suggest that combining commercial vaccines with rational booster immunization can effectively protect chickens against an nVarIBDV challenge.

Use of live attenuated recombinant Newcastle disease virus carrying avian paramyxovirus 2 HN and F protein genes to enhance immune responses against species A rotavirus VP6 protein

Numerous infectious diseases in cattle lead to reductions in body weight, milk production, and reproductive performance. Cattle are primarily vaccinated using inactivated vaccines due to their increased safety. However, inactivated vaccines generally result in weaker immunity compared with live attenuated vaccines, which may be insufficient in certain cases. Over the last few decades, there has been extensive research on the use of the Newcastle disease virus (NDV) as a live vaccine vector for economically significant livestock diseases. A single vaccination dose of NDV can sufficiently induce immunity; therefore, a booster vaccination dose is expected to yield limited induction of further immune response. We previously developed recombinant chimeric NDV (rNDV-2F2HN), in which its hemagglutinin-neuraminidase (HN) and fusion (F) proteins were replaced with those of avian paramyxovirus 2 (APMV-2). In vitro analysis revealed that rNDV-2F2HN expressing human interferon-gamma had potential as a cancer therapeutic tool, particularly for immunized individuals. In the present study, we constructed rNDV-2F2HN expressing the bovine rotavirus antigen VP6 (rNDV-2F2HN-VP6) and evaluated its immune response in mice previously immunized with NDV. Mice primarily inoculated with recombinant wild-type NDV expressing VP6 (rNDV-WT-VP6), followed by a booster inoculation of rNDV-2F2HN-VP6, showed a significantly stronger immune response than that in mice that received rNDV-WT-VP6 as both primary and booster inoculations. Therefore, our findings suggest that robust immunity could be obtained from the effects of chimeric rNDV-2F2HN expressing the same or a different antigen of a particular pathogen as a live attenuated vaccine vector.

Construction of and evaluation of the immune response to two recombinant pseudorabies viruses expressing the B119L and EP364R proteins of African swine fever virus.

African swine fever (ASF) is an infectious disease caused by ASF virus (ASFV), which is characterized by high infectivity, rapid onset of disease, and a high mortality rate. Outbreaks of ASFV have caused great economic losses to the global pig industry, and there is a need to develop safe and effective vaccines. In this study, two recombinant pseudorabies virus (PRV) strains, rGXGG-2016-ΔgI/ΔgE-EP364R and rGXGG-2016-ΔgI/ΔgE-B119L, expressing the EP364R and B119L protein, respectively, of ASFV, were constructed by homologous recombination technology. Western blotting and immunofluorescence analysis showed that these foreign proteins were expressed in cells infected with the recombinant strains. The strains showed good genetic stability and proliferative characteristics for 20 passages in BHK-21 cells. Both of these strains were immunogenic in mice, inducing the production of specific antibodies against the expressed ASFV proteins while providing protection against lethal challenge with PRV. Thus, the recombinant strains rGXGG-2016-ΔgI/ΔgE-EP364R and rGXGG-2016-ΔgI/ΔgE-B119L could be used as candidate vaccines for both ASFV and PRV. In addition, our study identifies two potential target genes for the development of safe and efficient ASFV vaccines, provides a reference for the construction of bivalent ASFV and PRV vaccines, and demonstrates the feasibility of developing a live ASFV vector vaccine.

A vesicular stomatitis virus-based African swine fever vaccine prototype effectively induced robust immune responses in mice following a single-dose immunization.

African swine fever (ASF) is a highly contagious hemorrhagic fever disease in pigs caused by African swine fever virus (ASFV). It is very difficult to control and prevent ASF outbreaks due to the absence of safe and effective vaccines. In order to develop a safe and effective ASF vaccine for the control and prevention of ASF, two ASFV recombinant vesicular stomatitis virus (VSV) live vector vaccine prototypes, containing the gene of p72, and a chimera of p30 and p54, were developed based on the replication-competent VSV, and named VSV-p72 and VSV-p35. The immune potency of VSV-p72 or VSV-p35 alone and in combination was evaluated in BALB/c mice via intramuscular and intranasal vaccination. The results indicated that whether administered alone or in combination, the two vaccine prototypes showed acceptable safety in mice and, more importantly, induced high-level specific antibodies against p72, p30, and p54 of ASFV and a strong cellular immune response 28 days after vaccination. The sera from mice vaccinated with the vaccine prototypes significantly inhibited ASFV from infecting porcine alveolar macrophages (PAMs) in vitro. Most notably, the immunized sera from a mixture of VSV-p35 and VSV-p72 inhibited ASFV from infecting PAMs, with an inhibition rate of up to 78.58%. Overall, our findings suggest that ASFV recombinant VSV live vector vaccine prototypes may become a promising candidate vaccine for the control and prevention of ASF.

Construction and Immunogenicity Evaluation of Recombinant Bacillus subtilis Expressing HA1 Protein of H9N2 Avian Influenza Virus.

The H9N2 subtype of the avian influenza virus (AIV) is one of the main subtypes of low pathogenic AIV, and it seriously affects the poultry breeding industry. Currently, vaccination is still one of China's main strategies for controlling H9N2 avian influenza. In this study, we selected MW548848.1 on the current popular main branch h9.4.2.5 as the reference strain, and we optimized the amino acid sequence of HA1 to make it suitable for expression in Bacillus subtilis. The B. subtilis expression vector showed good safety and stress resistance; therefore, this study constructed a recombinant B. subtilis expressing H9N2 HA1 protein and evaluated its immunogenicity in mice. The following results were obtained: the sIgA level of HA1 protein in small intestine fluid and the IgG level of PHT43-HA1/B. subtilis in serum were significantly improved (P < 0.01); PHT43-HA1/B. subtilis can cause a special immune response in mice; and cytokine detection interferon-gamma (IFN-γ) (P < 0.05) and Interleukin 2 (IL-2) (P < 0.01) expressions significantly increased. Additionally, the study found that PHT43-HA1/B. subtilis can alleviate the attack of H9N2 AIV in the spleen, lungs, and small intestine of mice. This study was the first to use an oral recombinant B. subtilis-HA1 vaccine candidate, and it provides theoretical data and technical reference for the creation of a new live vector vaccine against H9N2 AIV.

Bacillus subtilis expressing duck Tembusu virus E protein induces immune protection in ducklings

Duck Tembusu virus (DTMUV) is an infectious disease that emerged in China in 2010. It has caused serious economic losses to the poultry industry and may pose a threat to public health. We aimed to develop a new Bacillus subtilis (B. subtilis)-based oral vaccine to control DTMUV transmission among poultry; to this end, we constructed a B. subtilis strain that can secrete DTMUV E protein. Ducklings were orally immunized, and serum antibodies, mucosal antibodies, and splenic cytokines were detected. The results showed that, in addition to high levels of specific IgG, there were also high levels of specific secretory immunoglobulin A (sIgA) in ducklings orally treated with recombinant B. subtilis. In addition, the levels of IFN-γ, IL-2, IL-4, and IL-10 in spleens were significantly boosted by recombinant B. subtilis. Recombinant B. subtilis could effectively enhance ducklings resistance to DTMUV and significantly reduce viral load (p<0.01), along with pathological damage in the brain, heart, and spleen. This is the first study to apply a B. subtilis live-vector vaccine platform for DTMUV disease prevention and control, and our results suggest that B. subtilis expressing DTMUV E protein may be a candidate vaccine against DTMUV.

The effect of Toll-like receptor agonists on the immunogenicity of MVA-SARS-2-S vaccine after intranasal administration in mice.

Modified Vaccinia virus Ankara (MVA) represents a promising vaccine vector for respiratory administration to induce protective lung immunity including tertiary lymphoid structure, the bronchus-associated lymphoid tissue (BALT). However, MVA expressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein (MVA-SARS-2-S) required prime-boost administration to induce high titers of anti-Spike antibodies in serum and bronchoalveolar lavage (BAL). As the addition of adjuvants enables efficient tailoring of the immune responses even to live vaccines, we tested whether Toll-like receptor (TLR)-agonists affect immune responses induced by a single dose of intranasally applied MVA-SARS-2-S. We intranasally immunized C57BL/6 mice with MVA-SARS-2-S vaccine in the presence of either TLR3 agonist polyinosinic polycytidylic acid [poly(I:C)], TLR4 agonist bacterial lipopolysaccharide (LPS) from Escherichia coli, or TLR9 agonist CpG oligodeoxynucleotide (CpG ODN) 1826. At different time-points after immunization, we analyzed induced immune responses using flow cytometry, immunofluorescent microscopy, and ELISA. TLR agonists had profound effects on MVA-SARS-2-S-induced immune responses. At day 1 post intranasal application, the TLR4 agonist significantly affected MVA-induced activation of dendritic cells (DCs) within the draining bronchial lymph nodes, increasing the ratio of CD11b+CD86+ to CD103+CD86+ DCs. Nevertheless, the number of Spike-specific CD8+ T cells within the lungs at day 12 after vaccination was increased in mice that received MVA-SARS-2-S co-administered with TLR3 but not TLR4 agonists. TLR9 agonist did neither significantly affect MVA-induced DC activation nor the induction of Spike-specific CD8+ T cells but reduced both number and size of bronchus-associated lymphoid tissue. Surprisingly, the addition of all TLR agonists failed to boost the levels of Spike-specific antibodies in serum and bronchoalveolar lavage. Our study indicates a potential role of TLR-agonists as a tool to modulate immune responses to live vector vaccines. Particularly TLR3 agonists hold a promise to potentiate MVA-induced cellular immune responses. On the other hand, additional research is necessary to identify optimal combinations of agonists that could enhance MVA-induced humoral responses.

What Do We Know about Surface Proteins of Chicken Parasites Eimeria?

Poultry is the first source of animal protein for human consumption. In a changing world, this sector is facing new challenges, such as a projected increase in demand, higher standards of food quality and safety, and reduction of environmental impact. Chicken coccidiosis is a highly widespread enteric disease caused by Eimeria spp. which causes significant economic losses to the poultry industry worldwide; however, the impact on family poultry holders or backyard production-which plays a key role in food security in small communities and involves mainly rural women-has been little explored. Coccidiosis disease is controlled by good husbandry measures, chemoprophylaxis, and/or live vaccination. The first live vaccines against chicken coccidiosis were developed in the 1950s; however, after more than seven decades, none has reached the market. Current limitations on their use have led to research in next-generation vaccines based on recombinant or live-vectored vaccines. Next-generation vaccines are required to control this complex parasitic disease, and for this purpose, protective antigens need to be identified. In this review, we have scrutinised surface proteins identified so far in Eimeria spp. affecting chickens. Most of these surface proteins are anchored to the parasite membrane by a glycosylphosphatidylinositol (GPI) molecule. The biosynthesis of GPIs, as well as the role of currently identified surface proteins and interest as vaccine candidates has been summarised. The potential role of surface proteins in drug resistance and immune escape and how these could limit the efficacy of control strategies was also discussed.

Live Triple Gene-Deleted Pseudorabies Virus-Vectored Subunit PCV2b and CSFV Vaccine Undergoes an Abortive Replication Cycle in the TG Neurons following Latency Reactivation.

Like other alpha herpesviruses, pseudorabies virus (PRV) establishes lifelong latency in trigeminal ganglionic (TG) neurons. Upon stress, the latent viruses in the TG neurons reactivate and are transported anterograde from the neuron cell bodies to the nerve endings in the nasal mucosa, where they replicate and are discharged in the nasal and oral secretions. Consequently, the virus is transmitted to other naïve animals. This cycle of latency and reactivation continues until the animal dies or is slaughtered. We have constructed a PRV triple mutant virus (PRVtmv) and used it as a live subunit vaccine vector against porcine circovirus 2b (PCV2b) and classical swine fever virus (CSFV) (PRVtmv+). We compared the latency reactivation properties of PRVtmv+ with its parent wild-type (wt) Becker strain following intranasal infection. The results showed that PRV wt and PRVtmv+ established latency in the TG neurons. Based on nasal virus shedding, immediate early (infected cell protein 0; ICP0) and late genes, MCP (major capsid protein) and gC (glycoprotein C) transcriptions, and viral DNA copy numbers in the TGs of latently infected and dexamethasone (Dex)-treated pigs, both PRV wt and PRVtmv+ reactivated from latency. We noticed that PRV wt virus replicated productively in the terminally differentiated, postmitotic TG neurons, but PRVtmv+ failed to replicate and, therefore, there was no virus production in the TG. In addition, we found that only the PRV wt virus was shed in the nasal secretions following the Dex-induced reactivation. Our results demonstrated that the PRVtmv+ is safe as a live viral subunit vaccine vector without the possibility of productive replication in the TG upon reactivation from latency and without subsequent nasal virus shedding. This property of PRVtmv+ precludes the possibility of vaccine virus circulation in pigs and the risk of reversion to virulence.

A recombinant capripoxvirus expressing the F protein of peste des petits ruminants virus and the P12A3C of foot-and-mouth disease virus

BackgroundPeste des petits ruminants (PPR), foot-and-mouth disease (FMD) and sheep pox and goat pox are three important infectious diseases that infect goats, sheep and other small ruminants. It is well-known that the prevention of three diseases rely mainly on their individual vaccines. However, the vaccines have a variety of different disadvantages, such as short duration of immunity, increasing the number of vaccinations, and poor thermal stability. The purpose of this study is to construct a recombinant goat pox virus (rGPV) capable of expressing the F gene of PPRV and the P12A3C gene of FMDV as a live vector vaccine.ResultsThe IRES, FMDV P12A3C and PPRV F genes into the multi-cloning site of the universal transfer plasmid pTKfpgigp to construct a recombinant transfer plasmid pTKfpgigpFiP12A3C, and transfected GPV-infected lamb testis (LT) cells with liposomes and produced by homologous recombination Recombinant GPV (rGPV/PPRVF-FMDVP12A3C, rGPV). The rGPV was screened and purified by green florescence protein (GFP) and xanthine-guanine-phosphoribosyltransferase gene (gpt) of Escherichia coli as selective markers, and the expression of rGPV in LT cells was detected by RT-PCR and immunofluorescence techniques. The results showed that the virus strain rGPV/PPRVF-FMDVP12A3C containing FMDV P12A3C and PPRV F genes was obtained. The exogenous genes FMDV P12A3C and PPRV F contained in rGPV were normally transcribed and translated in LT cells, and the expression products could specifically react with PPRV and FMDV antiserum. Then, the rGPV was intradermally inoculated with goats, the animal experiments showed that rGPV/PPRVF-FMDVP12A3C could induce high levels of specific antibodies against GPV, PPRV and FMDV.ConclusionsThe constructed rGPV induced high levels of specific antibodies against GPV, PPRV and FMDV. The study provides a reference for “ one vaccine with multiple uses “ of GPV live vector vaccine.

Construction of a Lactobacillus plantarum-based claudin-3 targeting delivery system for the development of vaccines against Eimeria tenella.

Avian coccidiosis causes huge economic losses to the poultry industry worldwide and currently lacks effective live vector vaccines. Achieving efficient antigen delivery to mucosa-associated lymphoid tissue (MALT) is critical for improving the effectiveness of vaccines. Here, chicken claudin-3 (CLDN3), a tight junction protein expressed in MALT, was identified as a target, and the C-terminal region of Clostridium perfringens enterotoxin (C-CPE) was proven to bind to chicken CLDN3. Then, a CLDN3-targeting Lactobacillus plantarum NC8-expressing C-CPE surface display system (NC8/GFP-C-CPE) was constructed to successfully express the heterologous protein on the surface of L. plantarum. The colonization level of NC8/GFP-C-CPE was significantly increased compared to the non-targeting strain and could persist in the intestine for at least 72h. An oral vaccine strain expressing five EGF domains of Eimeria tenella microneme protein 8 (EtMIC8-EGF) (NC8/EtMIC8-EGF-C-CPE) was constructed to evaluate the protective efficacy against E. tenella infection. The results revealed that CLDN3-targeting L. plantarum induced stronger mucosal immunity in gut-associated lymphoid tissues (GALT) as well as humoral responses and conferred better protection in terms of parasite replication and pathology than the non-targeting strain. Overall, we successfully constructed a CLDN3-targeting L. plantarum NC8 surface display system characterized by MALT-targeting, which is an efficient antigen delivery system to confer enhanced protective efficacy in chickens against E. tenella infection.

Creation of poxvirus expressing foot-and-mouth and peste des petits ruminant disease virus proteins

Foot-and-mouth disease (FMD) and Peste des petits ruminant disease (PPR) are acute and severe infectious diseases of sheep and are listed as animal diseases for compulsory immunization. However, there is no dual vaccine to prevent these two diseases. The Modified Vaccinia virus Ankara strain (MVA) has been widely used in the construction of recombinant live vector vaccine because of its large capacity of foreign gene, wide hostrange, high safety, and immunogenicity. In this study, MVA-GFP recombinant virus skeleton was used to construct dual live vector vaccines against FMD and PPR. The recombinant plasmid pUC57-FMDV P1-2A3CPPRV FH was synthesized and transfected into MVA-GFP infected CEF cells for homologous recombination. The results showed that a recombinant virus without fluorescent labeling was obtained after multiple rounds of plaque screening. The recombinant virus successfully expressed the target proteins, and the empty capsid of FMDV could be observed by transmission electron microscope (TME), and the expression levels of foreign proteins (VP1 and VP3) detected by ELISA were like those detected in FMDV-infected cells. This study laidthe foundation for the successful construction of a live vector vaccine against FMD and PPR. • A recombinant MVA expressing FMDVP12A3C and PRRV HF proteins • Both the FMDV and PRRV proteins inserted into the virus were expressed • The proteins expressed by the recombinant poxvirus were assembled into VLPs.

Mucosal immune responses induced by oral administration of recombinant Lactococcus lactis expressing the S1 protein of PDCoV

Porcine deltacoronavirus is an evolving coronavirus that primarily infects the intestine and may lead to intestinal disease in piglets. Up to now, no commercial vaccination is readily accessible to protect against the spread of PDCoV. Lactococcus lactis has been shown to have good immune efficacy and safety and can be used as a genetically engineered vaccine to deliver antigens. In this research, we utilized L. lactis NZ9000 to provide the S1 protein orally and improved the delivery efficiency by connecting the M cell targeting ligand Co1 with the S1 protein of PDCoV in tandem to obtain the recombinant protein S1–Co1. We successfully constructed two recombinant strains capable of expressing PDCoV-S1 and PDCoV-S1-Co1 proteins (i.e., L. lactis NZ9000-S1 and L. lactis NZ9000-S1-Co1), and their immunogenic capacity was evaluated in mice. Our study shows that Lactococcus is an advantageous bacterial live vector vaccine and is anticipated as a potential PDCoV vaccination option.

Field efficacy of HVT-H9 vaccination after natural infection of H9N2 avian influenza virus in broilers

H9N2 subtype avian influenza virus (AIV) has been persistently circulating in China. It causes huge economic losses to the poultry industry and poses a great threat to public health. Previously, we constructed a turkey herpesvirus live vector vaccine candidate strain expressing an H9 gene, HVT-H9. Results showed that immunisation with HVT-H9 could provide good immunity in specific pathogen free (SPF) chickens. In this study, field-bred Arbour Acres plus (AA+) broilers were additionally immunised with HVT-H9 at one day of age. Then, broilers were naturally infected with H9N2 AIV. During the endemic period, death occurred in flocks without HVT-H9 immunisation and the mortality rate increased rapidly, forming a clear death wave. However, HVT-H9 vaccination prevented broiler mortality. Etiological tests and serological tests showed that broilers were positive for H9N2 AIV. Collectively, HVT-H9 immunisation provided good immunity for broilers in the field by inhibiting H9N2 virus infection and transmission.