A recombinant Newcastle disease virus (NDV) expressing infectious laryngotracheitis virus (ILTV) surface glycoprotein D protects against highly virulent ILTV and NDV challenges in chickens

Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). Currently, modified live ILTV vaccines are used to control ILT infections. However, the live ILTV vaccines can revert to virulence after bird-to-bird passage and are capable of establishing latent infections, suggesting the need to develop safer vaccines against ILT. We have evaluated the role of three major ILTV surface glycoproteins, namely, gB, gC, and gD in protection and immunity against ILTV infection in chickens. Using reverse genetics approach, three recombinant Newcastle disease viruses (rNDVs) designated rNDV gB, rNDV gC, and rNDV gD were generated, each expressing gB, gC, and gD, respectively, of ILTV. Chickens received two immunizations with rNDVs alone (gB, gC, and gD) or in combination (gB + gC, gB + gD, gC + gD, and gB + gC + gD). Immunization with rNDV gD induced detectable levels of neutralizing antibodies with the magnitude of response greater than the rest of the experimental groups including those vaccinated with commercially available vaccines. The birds immunized with rNDV gD showed complete protection against virulent ILTV challenge. The birds immunized with rNDV gC alone or multivalent vaccines consisting of combination of rNDVs displayed partial protection with minimal disease and reduced replication of challenge virus in trachea. Immunization with rNDV gB neither reduced the severity of the disease nor the replication of challenge virus in trachea. The superior protective efficacy of rNDV gD vaccine compared to rNDV gB or rNDV gC vaccine was attributed to the higher levels of envelope incorporation and infected cell surface expression of gD than gB or gC. Our results suggest that rNDV expressing gD is a safe and effective bivalent vaccine against NDV and ILTV.     

New baculovirus recombinants expressing Pseudorabies virus (PRV) glycoproteins protect mice against lethal challenge infection

The present study demonstrates the protective potential of novel baculovirus recombinants, which express the glycoproteins gB, gC, or gD of Pseudorabies virus (PRV; Alphaherpesvirus of swine) and additionally contain the glycoprotein G of Vesicular Stomatitis Virus (VSV-G) in the virion (Bac-G-PRV). To evaluate the protective capacity, mixtures of equal amounts of the PRV gB-, gC-, and gD-expressing baculoviruses were used for immunization. Three intramuscular immunizations with that Bac-G-PRV mixture could protect mice against a lethal PRV challenge infection. To achieve complete protection high titers of Bac-G-PRV and three immunizations were necessary. This immunization with Bac-G-PRV resulted in the induction of high titers of PRV-specific serum antibodies of the IgG2a subclass and of interferon (IFN)-gamma, indicating a Th1-type immune response. Moreover, splenocytes of immunized mice exhibited natural killer cell activity accompanied by the production of IFN-alpha and IFN-gamma. Collectively, the presented data demonstrate for the first time that co-expression of VSV-G in baculovirus recombinant vaccines can improve the induction of a protective immune response against foreign antigens.     

DNA vaccination of neonate piglets in the face of maternal immunity induces humoral memory and protection against a virulent pseudorabies virus challenge

DNA vaccination represents a unique opportunity to overcome the limitations of conventional vaccine strategy in early life in the face of maternal-derived immunity. We used the model of pseudorabies virus (PRV) infection in pigs to further explore the potential of DNA vaccination in piglets born to sows repeatedly vaccinated with a PRV inactivated vaccine. A single immunisation of 8-week-old piglets with a DNA vaccine expressing secreted forms of PRV gB, gC, and gD, triggered an active serological response, confirming that DNA vaccination can over-ride significant residual maternal-derived immunity. A clear anamnestic response was evidenced when a secondary DNA vaccination was performed at 11 weeks of age, suggesting that DNA vaccination, performed in the face of passive immunity, elicited a strong humoral memory. We subsequently explored the potential of DNA vaccination in neonate piglets (5-6 days of age) in the face of very high titres of maternal antibodies and demonstrated that very high titres of passive antibodies selectively inhibited serological responses but not the establishment of potent memory responses. Finally, we demonstrated that DNA vaccination provided protection against an infectious PRV challenge at the end of the fattening period (i.e. at approximately 5 months of age). Collectively, our results pave the way for a new flexible vaccination program, which could ensure uninterrupted protection of fattening pigs over their entire economical life under field conditions.     

Prime-boost immunization using DNA vaccine and recombinant Orf virus protects pigs against Pseudorabies virus (Herpes suid 1)

The present study demonstrates the protective potential of a novel prime-boost vaccination strategy of pigs against lethal Pseudorabies virus (PRV; Herpes suid 1) infection. Animals were primed with Sindbis virus-derived plasmids that express viral glycoproteins gC and gD (gC- and gD-pSIN) and subsequently booster immunized with Orf virus (ORFV; Parapoxvirus) recombinants expressing gC and gD (D1701-VrVgC and -VrVgD). The prime-boost vaccination induced strong humoral and cellular-like PRV-specific immune responses. All prime-boost vaccinated pigs survived the lethal challenge infection without PRV-specific clinical symptoms and presented excellent body weight loss attenuation. Most notably, nasal shedding of challenge virus was reduced by more than about 3log(10), clearly reducing the risk of infection of non-immunized pigs.     

CpG motif in ATCGAT hexamer improves DNA-vaccine efficiency against lethal Pseudorabies virus infection in pigs

This study evaluated the adjuvant activity of a porcine-specific type A CpG-containing oligodeoxynucleotide (CpG-ODN) in a DNA-vaccine against Pseudorabies virus (PrV) infection. This vaccine, composed of three independent plasmids encoding PrV gB, gC and gD glycoproteins, was injected alone or was co-injected with CpG-ODN or control GpC-ODN. CpG-ODN improved the PrV-specific humoral immune response and provided better clinical protection against lethal PrV-infection. The PrV-specific cell-mediated immune response seemed also to be increased.     

Effective protection of pigs against lethal Pseudorabies virus infection after a single injection of low-dose Sindbis-derived plasmids encoding PrV gB, gC and gD glycoproteins

This study compared protection of pigs against lethal Pseudorabies virus (PrV) infection induced by a single injection of various quantities of Sindbis virus-derived plasmids encoding PrV glycoproteins gB, gC and gD. Pigs were injected with 340, 68 or 13 microg of each plasmid. Few immune differences were observed after DNA injection and more importantly the pigs of the three groups were equally protected against virulent PrV infection. Single-shot injection of 13 microg of each PrV glycoprotein encoding Sindbis virus-derived plasmid is able to effectively protect pigs from PrV infection.     

Protection induced by intramuscular immunization with DNA vaccines of pseudorabies in mice, rabbits and piglets.

Glycoprotein gene gB, gC and gD of pseudorabies virus (PrV) strain Ea, which was isolated locally in Wuhan, were cloned from the viral genome DNA and expressed in vitro controlled by the major immediately-early promotor/enhancer of HCMV. In the presented paper, Balb/c mice, rabbits and piglets were vaccinated intramuscularly two times at 2-week interval with those eukaryotic expression plasmid pcDB, pcDC and pcDD, respectively. The animals injected with pcDB, pcDC, pcDD or mix DNA developed anti-PrV antibodies. Neutralizing antibody titers obtained 2-5log(2), 2 weeks after the second vaccination. Cellular immune responses were also detected by lymphoproliferation assay and cytotoxic T lymphocyte (CTL) activity assay in all groups vaccinated with DNA. Immune responses elicited by DNA vaccines provided protections with different degrees against lethal dose PrV challenge. In mice, protections induced by pcDC, pcDD or mix DNA were 100%, similar to that by inactivated vaccine. Protections were more than 50% induced by pcDC, pcDD or mix DNA in rabbits. Protections induced by pcDB were the lowest among DNA immunization in mice or rabbits. However, pcDB could elicit the higher cellular responses in rabbits or piglets. In piglets, body temperatures of animals injected with pcDB, pcDC, pcDD or mix DNA did not change significantly after challenge with 2x10(5) pfu of PrV strain Ea, and the means daily growth post-challenge of those animals were higher than those injected with inactivated vaccine or parental plasmid. Neither DNA vaccines nor inactivated vaccine could prevent or delay virus excretion after challenge. Our experiments in experimental animals and natural hosts suggested the efficiency and potential application of DNA vaccines for pseudorabies in pigs.     

Construction of a triple gene-deleted Chinese Pseudorabies virus variant and its efficacy study as a vaccine candidate on suckling piglets

New-emerging variants of Pseudorabies virus (PRV) compromise the protection provided by current vaccines and cause the death of all ages of vaccinated pigs since 2011. New vaccines based on current circulating PRV strain are needed to control the spread of disease since the variants are antigenically different from classical strains of virus. In this study, a TK/gE/gI triple gene-deleted PRV derived from current circulating field isolate was generated by using bacterial artificial chromosome techniques, and the rescued virus showed similar growth properties in vitro to its parent strain but reduced plaque size. To evaluate it as vaccine candidate, 9 day-old pigs were vaccinated and challenged with a virulent PRV variant. The results showed that vaccination can generate high level of protective gB-specific antibodies after vaccination and provide complete protection to the viral challenge. By contrast, the unvaccinated piglets all died within 6 days after viral challenge. Therefore, the TK/gE/gI triple gene-deleted PRV could be a promising vaccine candidate to control the wide spreading of PR variants in China.     

Bartha-k61 vaccine protects growing pigs against challenge with an emerging variant pseudorabies virus

Since late 2011, pseudorabies (PR) has resurfaced in many large pig farms, causing great economic loss for the swine industry in China. The PRV variant strain with high virulence and antigenic variation has been considered to be the main cause, and much attention has been focused on how to prevent and control the reoccurrence of this disease in China. In this study, two kinds of vaccination strategy were employed to evaluate the protective effects of Bartha-k61vaccine against both variant PRV (XJ5) and classical PRV (Ra) strain challenge. Humoral immunity response, clinical signs, survival rate, body weight, virus shedding and pathology were assessed in commercial pigs. The results showed that Bartha-k61vaccine, administered either once or twice, was effective against the PRV variant (XJ5) challenge, while no significant differences were observed between single and prime-boost vaccinated pigs. However, pigs vaccinated twice had better body weight gains than those vaccinated once, following challenge with the classical PRV strain (Ra) (p < 0.01). Therefore, the Bartha-k61 vaccine appears to be an effective vaccine to control the spread of PRV variants in China in the absence of new powerful candidate vaccines specific to these PRV strains.     

Vaccination of pigs against pseudorabies virus with plasmid DNA encoding glycoprotein D

We analysed the ability of a plasmid carrying the gene encoding glycoprotein D (gD) of pseudorabies virus (PRV) to induce humoral and cell-mediated immune responses and assessed the protection provided by PRV-gD DNA vaccination against challenge infection with PRV. Immunization with plasmid PRV-gD induced neutralizing antibodies and lymphocyte proliferative responses both in mice and pigs. Moreover, when challenged with virulent PRV six weeks following the last immunization, PRV-gD DNA vaccinated pigs excreted virus for a significantly shorter period and showed less clinical symptoms than pigs vaccinated with a control plasmid. Thus, in the target animal, DNA vaccination with PRV-gD DNA induces protective immunity against challenge infection.     

Immunogenicity and efficacy of recombinant subunit vaccines against phocid herpesvirus type 1

Phocid herpesvirus type 1 (PhHV-1) is an alpha-herpesvirus that causes significant morbidity and mortality among young and immunocompromised harbour seals (Phoca vitulina) and therefore represents a major problem for seal rehabilitation centres. Consequently, there is a need for a safe and effective PhHV-1 vaccine. We tested an ISCOM-based recombinant PhHV-1 gB vaccine alone (gB) or with the addition of recombinant PhHV-1 gD (gBD) for (i). immunogenicity and protective efficacy against feline herpesvirus (FHV) infection in cats and (ii). their immunogenicity in seals. The FHV-cat model was chosen based on the close antigenic relationship between PhHV-1 and FHV. Upon challenge, all vaccinated (gB and gBD) cats excreted significantly less FHV (P<0.01) and gBD vaccinated cats showed less weight loss (P=0.05) than the mock-vaccinated cats. However, adding gD to the gB vaccine did not result in significantly better protection. Based on these data, immunogenicity studies in seals under rehabilitation were performed with the gB vaccine only. To this end, gB vaccine was tested at two different doses (20 or 40 microg). PhHV-1 specific antibody titres and in vitro proliferative T cell responses were measured in all seals upon vaccination. No differences were observed in antibody titres between seals vaccinated with either 20 or 40 microgB, but the higher gB concentration did result in higher specific proliferative T cell responses (P<0.01). Based on the close antigenic relationship between PhHV-1 and FHV, the safety and efficacy data in the FHV-cat model, and the immunogenicity data in the vaccinated seals, the gB based vaccine is expected to be safe and effective in protecting against PhHV-1 related disease in harbour seals.     

Corneal macrophage infiltrates following ocular herpes simplex virus type 1 challenge vary in BALB/c mice vaccinated with different vaccines

Macrophage cell infiltrates in the cornea were examined following ocular herpes simplex virus type 1 (HSV-1) challenge of vaccinated BALB/c mice. Mice were vaccinated with individual HSV-1 glycoproteins, cocktails of different HSV-1 glycoproteins, or live avirulent HSV-1 (strain KOS). Cryostat sections of cornea were taken at different times after challenge and reacted with M1/70, F4/80, BM8, or MOMA-1 monoclonal antibodies. The pattern of macrophage responses in the cornea differed depending on the vaccine that was given prior to HSV-1 ocular challenge. No macrophage response was detected in mice vaccinated with the highly protective 5gPs consisting of the five glycoproteins gB, gC, gD, gE, and gI. In contrast, mock vaccinated mice and mice vaccinated with gK, which is known to exacerbate HSV-1 induced eye disease, had high sustained macrophage responses. Mice vaccinated with 7gPs (5gPs+gG and gH) had moderate levels of macrophages. It appeared that (1) the most effective vaccines induced no detectable infiltrating macrophages in the eyes, while the least efficacious vaccines had very high levels of infiltrating macrophages; (2) presence of CD11b(+) cells in the cornea appeared to correlate with enhanced blepharitis, but did not appear to affect corneal scarring; and (3) presence of F4/80(+) cells in the cornea tended to correlate with increased corneal scarring.     

Prime-boost strategies combining DNA and inactivated vaccines confer high immunity and protection in cattle against bovine herpesvirus-1

DNA vaccines have frequently been associated with poor efficacy in large animals. In the present study, one administration of an inactivated marker vaccine to cattle considerably boosted both humoral and cellular arms of the immune response primed with Bovine herpesvirus-1 (BoHV-1) DNA vaccines encoding glycoprotein D (gD) or gC+gD. Calves vaccinated according to the DNA prime-inactivated boost also showed significantly enhanced virological protection as compared to controls. The 4-logarithms reduction of virus shedding observed in primed-boosted animals was comparable to the one previously reported in calves immunized twice with marker vaccines. Intradermal immunization of cattle with DNA vaccines promoted a Th2-biased immune response but also primed a cellular component that was further boosted by the inactivated vaccine. Individual IgG2 titers of vaccinated calves were significantly correlated to IFN-gamma production. The immunization protocol described in the present study demonstrates the complementarity between DNA and conventional marker vaccines.     

Antibody and cellular immune responses of naïve mares to repeated vaccination with an inactivated equine herpesvirus vaccine

Equine herpesvirus type 1 (EHV-1) continues to cause severe outbreaks of abortions or myeloencephalopathy in horses despite widely used vaccination. The aim of this work was to determine the effects of frequent vaccination with an inactivated EHV vaccine on immune development in horses. Fifteen EHV-1 naïve mares were vaccinated a total of 5 times over a period of 8 months with intervals of 20, 60, 90 and 60 days between vaccine administrations. Total antibody and antibody isotype responses were evaluated with a new sensitive EHV-1 Multiplex assay to glycoprotein C (gC) and gD for up to 14 months after initial vaccination. Antibodies peaked after the first two vaccine doses and then declined despite a third administration of the vaccine. The fourth vaccine dose was given at 6 months and the gC and gD antibody titers increased again. Mixed responses with increasing gC but decreasing gD antibody values were observed after the fifth vaccination at 8 months. IgG4/7 isotype responses mimicked the total Ig antibody production to vaccination most closely. Vaccination also induced short-lasting IgG1 antibodies to gC, but not to gD. EHV-1-specific cellular immunity induced by vaccination developed slower than antibodies, was dominated by IFN-γ producing T-helper 1 (Th1) cells, and was significantly increased compared to pre-vaccination values after administration of 3 vaccine doses. Decreased IFN-γ production and reduced Th1-cell induction were also observed after the second and fourth vaccination. Overall, repeated EHV vaccine administration did not always result in increasing immunity. The adverse effects on antibody and cellular immunity that were observed here when the EHV vaccine was given in short intervals might in part explain why EHV-1 outbreaks are observed worldwide despite widely used vaccination. The findings warrant further evaluation of immune responses to EHV vaccines to optimize vaccination protocols for different vaccines and horse groups at risk.     

Comparison of monovalent glycoprotein B with bivalent gB/pp65 (GP83) vaccine for congenital cytomegalovirus infection in a guinea pig model: Inclusion of GP83 reduces gB antibody response but both vaccine approaches provide equivalent protection against pup mortality

Cytomegalovirus (CMV) subunit vaccine candidates include glycoprotein B (gB), and phosphoprotein ppUL83 (pp65). Using a guinea pig cytomegalovirus (GPCMV) model, this study compared immunogenicity, pregnancy outcome, and congenital viral infection following pre-pregnancy immunization with a three-dose series of modified vaccinia virus Ankara (MVA)-vectored vaccines consisting either of gB administered alone, or simultaneously with a pp65 homolog (GP83)-expressing vaccine. Vaccinated and control dams were challenged at midgestation with salivary gland-adapted GPCMV. Comparisons included ELISA and neutralizing antibody responses, maternal viral load, pup mortality, and congenital infection rates. Strikingly, ELISA and neutralization titers were significantly lower in the gB/GP83 combined vaccine group than in the gB group. However, both vaccines protected against pup mortality (63.2% in controls vs. 11.4% and 13.9% in gB and gB/GP83 combination groups, respectively; p < 0.0001). Reductions in pup viral load were noted for both vaccine groups compared to control, but preconception vaccination resulted in a significant reduction in GPCMV transmission only in the monovalent gB group (26/44, 59% v. 27/34, 79% in controls; p < 0.05). We conclude that, using the MVA platform, the addition of GP83 to a gB subunit vaccine interferes with antibody responses and diminishes protection against congenital GPCMV infection, but does not decrease protection against pup mortality.     

Pre-existing immunity to cytomegalovirus in macaques influences human CMV vaccine responses in preclinical models

Development of a human cytomegalovirus (HCMV) vaccine is a Tier 1 priority by the National Institutes of Medicine, as HCMV is the most common congenital infection globally and most frequent infectious complication in transplant patients. Relevant preclinical non-human primate models used for testing HCMV vaccine immunogenicity are rhesus and cynomolgous monkeys. However, a complication in using these models is that species-specific CMV variants are endemic in non-human primate breeding colonies. We hypothesize that natural immunity to species-specific CMV in rhesus and cynomolgous monkeys impacts HCMV vaccine immunogenicity and may interfere with our ability to fully interpret vaccine immunogenicity. A modified mRNA vaccine encoding HCMV glycoprotein (gB) and the pentameric complex (PC) packaged in lipid nanoparticles (LNP) was delivered intramuscularly to groups of cynomolgous (n = 16, CyCMV-seropositive) and rhesus macaques (n = 24, RhCMV-seropositive). High pre-vaccination IgG binding responses to HCMV gB were present in both species, but pre-vaccination binding responses to PC were mostly present in rhesus macaques. Yet, at least a log increase in both PC and gB-specific plasma IgG levels was detected post-second HCMV mRNA vaccination in both species. Both species responded with high epithelial cell neutralizing antibody responses at 4 weeks post second HCMV mRNA vaccination, but limited fibroblast neutralizing antibodies. HCMV gB + PC mRNA/LNP vaccine also elicited IgG binding responses to cell-associated gB, an identified immune correlate of protection, in both species after the second vaccination, and there was a moderately strong direct correlation between this pre- and post-vaccination response in rhesus macaques. Based on the correlation between pre-existing and post-vaccine gB-specific binding responses in rhesus macaques, we conclude that species-specific CMV variant-specific antibody responses contribute to antibody responses to HCMV vaccination in primate models, indicating that pre-existing immunity must be taken into account in non-human primate preclinical models and will impact immunogenicity of HCMV vaccines seropositive human vaccinees.     

Antibody responses to crucial functional epitopes as a novel approach to assess immunogenicity of vaccine adjuvants

We are interested in developing a vaccine that prevents genital herpes. Adjuvants have a major impact on vaccine immunogenicity. We compared two adjuvants, an experimental Merck Sharp & Dohme lipid nanoparticle (LNP) adjuvant, LNP-2, with CpG oligonucleotide combined with alum for immunogenicity in mice when administered with herpes simplex virus type 2 (HSV-2) glycoproteins C, D and E (gC2, gD2, gE2). The immunogens are intended to produce neutralizing antibodies to gC2 and gD2, antibodies to gD2 and gE2 that block cell-to-cell spread, and antibodies to gE2 and gC2 that block immune evasion from antibody and complement, respectively. Overall, CpG/alum was better at producing serum and vaginal IgG binding antibodies, neutralizing antibodies, antibodies that block virus spread from cell-to-cell, and antibodies that block immune evasion domains on gC2. We used a novel high throughput biosensor assay to further assess differences in immunogenicity by mapping antibody responses to seven crucial epitopes on gD2 involved in virus entry or cell-to-cell spread. We found striking differences between CpG/alum and LNP-2. Mice immunized with gD2 CpG/alum produced higher titers of antibodies than LNP-2 to six of seven crucial epitopes and produced antibodies to more crucial epitopes than LNP-2. Measuring epitope-specific antibodies helped to define mechanisms by which CpG/alum outperformed LNP-2 and is a valuable technique to compare adjuvants.     

Immunogenicity and protective efficacy of a gE, gG and US2 gene-deleted bovine herpesvirus-1 (BHV-1) vaccine.

The efficacy and safety of a gene-deleted bovine herpesvirus-1 (BHV-1) vaccine was determined in a bovine herpesvirus challenge trial in calves. Three different doses of the vaccine were administered intramuscularly at 10(5), 10(6) and 10(7) PFU/ml and compared to a commercial vaccine and non vaccinated control calves. Challenge was performed by intranasal aerosolization with the Cooper strain of BHV-1 (3 x 10(4) PFU/ml). The non-vaccinated calves shed significantly (P < 0.05) more virus than all other groups on days 4, 8 and 10 post challenge. By day 14 post challenge, antibody titers for BHV-1 of calves vaccinated with 10(7) PFU/ml were significantly (P < 0.05) higher than the commercial or non-vaccinated calves. Clinical scores of non-vaccinated calves were significantly (P < 0.05) higher than all other groups on days 4-14 post challenge. With both radioimmunoprecipitation and competitive enzyme-linked immunosorbent assays (C-ELISA), calves in the gene-deleted vaccine groups mounted comparable specific responses against gB, gC and gD post vaccination as calves in the commercial vaccine group, but in a dose dependent manner. These data suggest that the gene-deleted BHV-1 vaccine tested may be used as an effective vaccine in controlling BHV-1 infections.     

A simple selection system for construction of recombinant gD-negative pseudorabies virus as a vaccine vector.

We describe a simple, efficient two-step method for construction of glycoprotein D (gD)-negative pseudorabies virus (PrV) carrying transgenes inserted in place of the gD gene. The first step was the use of the thymidine kinase (TK) gene of herpes simplex virus (HSV) for insertional inactivation of the gD gene in a PrV mutant deficient in both TK and glycoprotein E (gE). The gD-negative, HSV-TK-positive mutant could be selected in HAT medium. The second step was substitution of HSV-TK with other genes of interest. The resultant gD/gE/TK-negative mutant was easily isolated by acyclovir selection. The expression of the transgene was detectable in vivo and the antibody responses against both inserted antigens and PrV were induced. The protective efficacy of the gD/gE/TK-negative PrV against lethal PrV challenge was also demonstrated. This PrV mutant carrying immunogenic proteins from unrelated porcine pathogens may be tested as a multivalent vaccine candidate for swine.