A candidate phocid herpesvirus vaccine that provides protection against feline herpesvirus infection.

Phocid herpesvirus type 1 (PhHV-1) causes significant morbidity and mortality among young and immunocompromised harbour seals. Therefore, the availability of an effective PhHV-1 vaccine would be of importance for orphanages and seal rehabilitation centres. Since possibilities to test PhHV-1 candidate vaccines in the target species are limited, a suitable animal model is needed. Given the close genetic and antigenic relationships between PhHV-1 and feline herpesvirus (FHV), the FHV cat system could be considered to test candidate PhHV-1 vaccines. Here we have tested a PhHV-1 based ISCOM vaccine for its protective efficacy against FHV infection in cats. To this end, three groups of cats were vaccinated thrice with ISCOM adjuvanted PhHV-1, FHV, and mock vaccines, respectively. One month after the last vaccination, all cats were challenged with a virulent FHV strain. All PhHV-1 and FHV vaccinated cats were protected from developing severe disease and excreted significantly less FHV than the mock vaccinated cats.     

Purification of phocid herpesvirus type 1 glycoproteins B and D and pilot studies of immunogenicity in mice

In the process of developing a subunit vaccine against phocid herpesvirus type 1, we have cloned and expressed the glycoproteins B and D (gB and gD) of phicid herpesvirus type 1, using an eukaryotic baculovirus expression system. To establish the proof of concept, candidate iscom vaccines based on these affinity-purified proteins either alone or in combination, were tested for their immunogenicity in BALB/C mice. Mice immunised with a combination of gB and gD developed higher antibody and proliferative T cell responses against PhHV-1 than those immunised with gB or gD alone. The corresponding antibody and T cell proliferative responses were higher against PhHV-1 than against FHV. These data favour further testing of these candidate vaccines based on gB and gD in the FHV-cat model.     

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.     

Canine distemper virus ISCOMs induce protection in harbour seals (Phoca vitulina) against phocid distemper but still allow subsequent infection with phocid distemper virus-1.

A candidate canine distemper virus (CDV) ISCOM vaccine has been shown to be effective in protecting harbour seals (Phoca vitulina) from phocid distemper in 1988. However, of the 35 harbour seals receiving this vaccine upon admission to a seal rehabilitation and research centre (Pieterburen, The Netherlands) in 1989, six developed mild inflammatory symptoms of the respiratory tract. Phocid distemper virus-1 (PDV-1) could be isolated from three of these animals. This indicates that the vaccine affords protection from phocid distemper, but may still allow PDV-1 infection of the respiratory tract. Contacts with non-vaccinated seals should then be prevented until no more virus is excreted. It is speculated that this PDV-1 infection of the respiratory tract in CDV-ISCOM vaccinated seals is followed by a lifelong immunity.     

Vaccination of harbour seals (Phoca vitulina) against phocid distemper with two different inactivated canine distemper virus (CDV) vaccines

Two inactivated canine distemper virus (CDV) vaccines—an adjuvanted whole inactivated virus and a subunit ISCOM preparation—were tested for their ability to induce protective immunity in harbour seals (Phoca vitulina) against phocid distemper, a disease that recently killed> 17 000 harbour seals in the North and Baltic seas, and was shown to be caused by infection with a newly discovered morbillivirus, which is antigenically closely related to CDV. Four CDV seronegative harbour seals were vaccinated three times with the whole-virus vaccine, two with the ISCOM subunit vaccine and two were sham-vaccinated with an antigen-free preparation. Ten days after the last vaccination, when all six vaccinated animals had developed CDV neutralizing antibody titres ranging from 300 to 3000, all eight animals were challenged by the oculonasal and the peritoneal routes, with an organ suspension from dead seals. None of the six vaccinated animals developed clinical signs. The two sham-vaccinated seals died on days 14 and 18, respectively, after having shown a body temperature rise, respiratory symptoms and weight loss. In organs from both dead animals morbillivirus antigen was demonstrated with an enzyme-linked immunosorbent assay and an immunofluorescence assay. One of these two animals had developed a low titre of CDV-specific antibodies just before death. These data clearly indicate that seals can be protected from fatal challenge with the phocid distemper virus (PDV), by vaccination with certain inactivated CDV vaccines. They also reconfirm that infection with PDV should be considered the primary cause of the recent epizootic in seals.     

Vaccination of harbour seals (Phoca vitulina) against phocid distemper with two different inactivated canine distemper virus (CDV) vaccines

Two inactivated canine distemper virus (CDV) vaccines—an adjuvanted whole inactivated virus and a subunit ISCOM preparation—were tested for their ability to induce protective immunity in harbour seals (Phoca vitulina) against phocid distemper, a disease that recently killed > 17 000 harbour seals in the North and Baltic seas, and was shown to be caused by infection with a newly discovered morbillivirus, which is antigenically closely related to CDV. Four CDV seronegative harbour seals were vaccinated three times with the whole-virus vaccine, two with the ISCOM subunit vaccine and two were sham-vaccinated with an antigen-free preparation. Ten days after the last vaccination, when all six vaccinated animals had developed CDV neutralizing antibody titres ranging from 300 to 3000, all eight animals were challenged by the oculonasal and the peritoneal routes, with an organ suspension from dead seals. None of the six vaccinated animals developed clinical signs. The two sham-vaccinated seals died on days 14 and 18, respectively, after having shown a body temperature rise, respiratory symptoms and weight loss. In organs from both dead animals morbillivirus antigen was demonstrated with an enzyme-linked immunosorbent assay and an immunofluorescence assay. One of these two animals had developed a low titre of CDV-specific antibodies just before death. These data clearly indicate that seals can be protected from fatal challenge with the phocid distemper virus (PDV), by vaccination with certain inactivated CDV vaccines. They also reconfirm that infection with PDV should be considered the primary cause of the recent epizootic in seals.     

Immune responses of mice against recombinant bovine herpesvirus 5 glycoprotein D

Glycoprotein D (gD) is essential for attachment and penetration of Bovine herpesvirus 5 (BoHV-5) into permissive cells, and is a major target of the host immune system, inducing strong humoral and cellular immune responses. The aim of this study was to evaluate in mice the immunogenicity of recombinant BoHV-5 gD (rgD5) expressed in Pichia pastoris. Vaccines formulated with rgD5 alone or adjuvanted with Montanide 50 ISA V2; Emulsigen or Emulsigen-DDA was administered intramuscularly or subcutaneously. Almost all formulations stimulated a humoral immune response after the first inoculation. The only exception was observed when the rgD5 was administered subcutaneously without adjuvant, in this case, the antibodies were observed after three doses. Higher titers of neutralizing antibodies were obtained with the three oil-based adjuvant formulations when compared to non-adjuvanted vaccine formulations. The rgD5 vaccine stimulated high mRNA expression levels of Th1 (INF-γ) and pro-inflammatory cytokines (IL-17, GM-CSF). The results demonstrated that the recombinant gD from BoHV-5 conserved important epitopes for viral neutralization from native BoHV-5 gD and was able to elicit mixed Th1/Th2 immune response in mice.     

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.     

Native herpes simplex virus glycoprotein D vaccine: immunogenicity and protection in animal models.

Mice, guinea pigs, and rhesus monkeys were immunized with immunoaffinity-purified native glycoprotein D (gD) derived from herpes simplex virus type 1 (HSV1). The native glycoprotein has evoked significant in vivo responses even at low doses. Thus, mice immunized with doses as low as 1 microgram were significantly protected from the morbidity and mortality of lethal HSV2 challenge and from establishment of latent HSV2 infection. Protection was dose-related and correlated with prechallenge serum neutralizing antibody titres to HSV. Similarly, immunized guinea-pigs demonstrated significant reductions in the frequency, severity and duration of genital lesions induced by HSV2 vaginal challenge. In long term immunogenicity studies, immunized rhesus monkeys exhibited significant serum neutralizing antibody responses to both HSV1 and HSV2. In vitro stimulation of monkey peripheral blood leucocytes with purified gD resulted in a significant cellular proliferative response. The results obtained in these animal models with a gD subunit vaccine provide an appropriate foundation for the initiation of human studies.     

A single dose glycoprotein D-based subunit vaccine against pseudorabies virus infection

Pseudorabies Virus (PRV) is the causative agent of Pseudorabies (PR), also known as Aujeszky’s Disease, one of the most important infectious diseases in swine, resulting in huge economic losses to the swine industry globally. The emergence of mutant PRV strains after 2011 resulted in a sharp decrease in the efficacy of available commercial vaccines. To develop a more effective vaccine that can prevent the spread of PRV, glycoprotein B (gB), glycoprotein C (gC) and glycoprotein D (gD) from recent PRV isolates were expressed in a baculovirus system and their protective efficacy was tested in mice and piglets. Neutralizing antibody titers (NAs) in mice vaccinated with gB, gC and gD peaked at 28 days after immunization and then slowly declined. NAs in the mice immunized with gD were remarkably higher than other groups. After a lethal challenge of 5 LD₅₀ with mutant PRV-HNLH strain, the survival rates of gB and gD were 100% and 87.5% respectively, which was significantly higher than gC group (50%). Piglets vaccinated with the gD and gB + D vaccines developed the highest NAs 7 days post immunization. No piglets in these two groups exhibited clinical symptoms, high body temperature or virus shedding following challenge with 10^6.6 TCID₅₀ with the mutant PRV-HNLH strain. Histopathology and immunohistochemistry showed remarkably reduced pathological damage and viral loads in gD and gB + D groups. Furthermore, the duration of the NAs induced by gD vaccine could maintain as long as four months after a single dose. The current study indicates that a gD-based vaccine could be developed for the efficient control of PRV.     

A prime-boost immunization strategy with DNA and recombinant baculovirus-expressed protein enhances protective immunogenicity of glycoprotein D of equine herpesvirus 1 in naïve and infection-primed mice

The immunogenicity and protective efficacy afforded by intramuscular inoculation of plasmid DNA encoding equine herpesvirus 1 (EHV-1) glycoprotein D (gD) followed by EHV-1 gD expressed by a recombinant baculovirus was assessed in a murine model of EHV-1 respiratory infection. Compared with mice inoculated with DNA or protein only, mice inoculated with the combination of gD DNA and protein had enhanced ELISA and neutralizing antibody titres to EHV-1 and had accelerated clearance of virus from lungs following challenge infection. The enhanced protective effects of this consecutive immunization were also evident in mice which had a previous infection with EHV-1 and had pre-existing antibodies. The T-helper 1 (Th1) type of immune response induced by EHV-1 gD DNA was maintained after the protein boost, despite the gD protein alone appearing to direct a Th2 response.     

Parasite prevalence in free-ranging farm cats, Felis silvestris catus.

No animals tested were positive for feline leukemia virus antigen and Chlamydia psittaci antibodies, but all were positive for antibodies to feline calicivirus (FCV), feline herpesvirus 1 (FHV1) and rotavirus. They had antibodies to feline parvovirus (96%), feline coronavirus (84% and cowpox virus (2%). Antibody to feline immunodeficiency virus (FIV) was found in 53% of animals, which were less likely to be infected with Haemobartonella felis, and had higher FHV antibody titres than cats without FIV. FCV was isolated from 51% cats and FHV1 and feline reovirus each from 4%. H. felis was present in 42% of animals, and antibody to Toxoplasma gondii in 62%. Clinical abnormality had a significant association with FIV and feline calicivirus infections, but sex, age, social status and feeding group had no significant association with prevalence of any parasites. Toxocara cati and Toxascaris leonina eggs were found, respectively, in 91% and 82% of animals tested.     

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.     

Herpesvirus infections in seals: a summary of present knowledge.

This review summarises the occurrence of herpesvirus infections in pinnipeds and data from investigations carried out by the authors. These data demonstrate that herpesvirus isolates collected from harbour seals (Phoca vitulina Linnaeus, 1758) during the 1988 seal epidemic were almost indistinguishable from feline herpesvirus 1 and that Antarctic Weddell's seals (Leptonychotes weddelli Lesson, 1826) carry antibodies against seal herpesvirus from the north-western Europe seal epidemic. The significance of herpesvirus infections in seals is discussed.     

Humoral immunity targeting site I of antigenic domain 2 of glycoprotein B upon immunization with different cytomegalovirus candidate vaccines

Glycoprotein B (gB) is a major component in several vaccines that are under development for prevention of disease by cytomegalovirus. It contains multiple determinants that are targets for neutralizing antibodies. One of them is site I of antigenic domain 2 (AD-2). The epitope, defined by short peptides, is quite conserved between different isolates. However, it is poorly immunogenic in natural infection. In this study we investigated the extent to which different vaccines, attenuated live Towne vaccine with or without priming with a canarypox virus coding for gB, or a recombinant gB vaccine adjuvanted with MF59, induced antibodies to this epitope. As in natural infection only a fraction of all subjects developed antibody responses against site I of AD-2 following vaccination. We suggest that strategies that enhance immunogenicity of this epitope will improve vaccine efficacy.     

Efficacy of a B virus gD DNA vaccine for induction of humoral and cellular immune responses in Japanese macaques

It is desirable to prevent dissemination of B virus (BV) in macaque colonies because transmission of BV to humans causes deadly encephalomyelitis. Vaccination of monkeys is one method that could confine spread of BV within macaque colonies. Availability of a BV DNA vaccine for use in macaques would eliminate the risk of working with infectious BV. Toward this end, we constructed a plasmid expressing the BV glycoprotein D (gD). Immunogenicity of this construct as a DNA vaccine was assessed in adult Japanese macaques by four intracutaneous injections at a dose of 500 microg per head. Results of enzyme-linked immunosorbent assay (ELISA) using a recombinant herpes simplex virus type 1 (HSV1) gD, a homologue of BV gD, showed that significant levels of antibody was induced in all vaccinated animals following each booster injection. Western blot of sera from vaccinated macaques confirmed the specific recognition of authentic BV gD. Immune sera were also demonstrated to contain neutralizing activity against infectious BV. Weak lymphoproliferative responses were also observed in vaccinated macaques using recombinant HSV1 gD as a stimulating antigen and flow cytometry analysis of one individual revealed the presence of HSV1 gD-responsive effector T cells. Thus, the BV gD DNA vaccine was demonstrated to induce both humoral and cellular immune responses in macaques which recognized BV gD.     

Safety and immunogenicity of three lots of meningococcal serogroup C conjugate vaccine administered at 2, 3 and 4 months of age

The reactogenicity and immunogenicity of meningococcal serogroup C conjugate (MenC) vaccine was assessed in 322 infants vaccinated at 2, 3, and 4 months of age, with concomitant administration of mixed diphtheria-tetanus-whole-cell pertussis vaccine and Haemophilus influenzae type b conjugate vaccine (DTwP-Hib) and oral polio vaccine. All infants in whom post-vaccination meningococcal C anticapsular IgG levels were assayed (n = 265) attained > or = 2 microg ml(-1). Serum bactericidal titres were assayed for a proportion of subjects (n = 171), 98% of whom obtained a reciprocal titres > or = 8. Local reactions were less frequent at the MenC injection site than at the DTP-Hib site. Systemic events were frequent, but consistent with established DTwP-Hib experience. The study demonstrates that MenC vaccine is immunogenic and well tolerated in infants at manufacturing scale production levels.     

Effects of herpes simplex virus type 2 glycoprotein vaccines and CLDC adjuvant on genital herpes infection in the guinea pig

Genital herpes simplex virus (HSV) infections are common but results from vaccine trials with HSV-2 glycoprotein D (gD) have been disappointing. We therefore compared a similar HSV gD2 vaccine, to a further truncated gD2 vaccine, to a vaccine with gD2 plus gB2 and gH2/gL2 and to a vaccine with only gB2 and gH2/gL2 in a guinea pig model of genital herpes. All vaccines were administered with cationic liposome-DNA complexes (CLDC) as an adjuvant. All vaccines significantly decreased the severity of acute genital disease and vaginal virus replication compared to the placebo group. The majority of animals in all groups developed at least one episode of recurrent disease but the frequency of recurrent disease was significantly reduced by each vaccine compared to placebo. No vaccine was significantly more protective than gD2 alone for any of the parameters described above. No vaccine decreased recurrent virus shedding. When protection against acute infection of dorsal root ganglia and the spinal cord was evaluated all vaccines decreased the per cent of animal with detectable virus and the quantity of virus but again no vaccine was significantly more protective than another. Improvements in HSV-2 vaccines may require inclusion of more T cell targets, more potent adjuvants or live virus vaccines.     

Evaluation of safety and immunogenicity of feline vaccines with reduced volume

A non adjuvanted vaccine against feline herpesvirus, feline calicivirus, feline panleucopenia and feline leukemia has been formulated in reduced volume (0.5 ml) with the same antigen content as the conventional 1 ml presentation. This paper reports studies evaluating the safety and the immunogenicity of this reduced volume vaccine in comparison with the conventional volume vaccine. The safety of both vaccines was evaluated in a small sized laboratory trial. It was further tested in a randomized controlled field trial on a total of 398 cats. Immediate and delayed local and systemic adverse events were monitored after vaccination. The immunogenicity of each vaccine was also checked by serological antibody responses against the vaccines antigens during the laboratory trial.These studies showed that the 0.5 ml vaccine was well tolerated in cats, inducing less local events, while keeping the same immunogenicity as the corresponding 1 ml vaccine. Reducing the volume of the vaccine is a way to improve the convenience of administration and to help following vaccination guidelines with the aim of reducing the incidence of adverse events following vaccination.