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Highlights ► Mammalian review. ► Important veterinary viral immunodiseases. ► Immunoprophylaxis. ► Other control measures. ► Current status of vaccine research., This paper offers an overview of important veterinary viral diseases of mammals stemming from aberrant immune response. Diseases reviewed comprise those due to lentiviruses of equine infectious anaemia, visna/maedi and caprine arthritis encephalitis and feline immunodeficiency. Diseases caused by viruses of feline infectious peritonitis, feline leukaemia, canine distemper and aquatic counterparts, Aleutian disease and malignant catarrhal fever. We also consider prospects of immunoprophylaxis for the diseases and briefly other control measures. It should be realised that the outlook for effective vaccines for many of the diseases is remote. This paper describes the current status of vaccine research and the difficulties encountered during their development.

Infection with human influenza virus leads to serious respiratory disease. Vaccination is the most common and effective prophylactic measure to prevent influenza. Influenza vaccine manufacturing and release is controlled by the correct determination of the potency-defining haemagglutinin (HA) content. This determination is historically done by single radial immunodiffusion (SRID), which utilizes a statistical slope-ratio model to estimate the actual HA content. In this paper we describe the development and qualification of a parallel line model for analysis of HA quantification by SRID in cell culture-derived whole virus final monovalent and trivalent influenza vaccines. We evaluated plate layout, sample randomization, and validity of data and statistical model. The parallel line model was shown to be robust and reproducible. The precision studies for HA content demonstrated 3.8-5.0% repeatability and 3.8%-7.9% intermediate precision. Furthermore, system suitability criteria were developed to guarantee long-term stability of this assay in a regulated production environment. SRID is fraught with methodological and logistical difficulties and the determination of the HA content requires the acceptance of new and modern release assays, but until that moment, the described parallel line model represents a significant and robust update for the current global influenza vaccine release assay. (C) 2011 Elsevier Ltd. All rights reserved.

Recently we reported that reacetylation of N,N,N-trimethyl chitosan (TMC) reduced the adjuvant effect of TMC in mice after intranasal (i.n.) administration of whole inactivated influenza virus (WIV) vaccine. The aim of the present study was to elucidate the mechanism of this lack of adjuvanticity. Reacetylated TMC (TMC-RA, degree of acetylation 54%) was compared with TMC (degree of acetylation 17%) at six potentially critical steps in the induction of an immune response after i.n. administration in mice. TMC-RA was degraded in a nasal wash to a slightly larger extent than TMC. The local i.n. distribution and nasal clearance of WIV were similar for both TMC types. Fluorescently labeled WIV was taken up more efficiently by Calu-3 cells when formulated with TMC-RA compared to TMC and both TMCs significantly reduced transport of WIV over a Calu-3 monolayer. Murine bone-marrow derived dendritic cell activation was similar for plain WIV, and WIV formulated with TMC-RA or TMC. The inferior adjuvant effect in mice of TMC-RA over that of TMC might be caused by a slightly lower stability of TMC-RA-WIV in the nasal cavity, rather than by any of the other factors studied in this paper. © 2011 Elsevier B.V. All rights reserved.

The cold-adapted, temperature sensitive and attenuated influenza master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/ 60/69 were used to generate the vaccine viruses to be included in live attenuated influenza vaccine. These vaccine viruses typically are 6:2 reassortant viruses containing the surface antigens hemagglutinin and neuraminidase of current wild type influenza A and influenza B viruses with the gene segments encoding the internal viral proteins, and conferring the cold-adapted, temperature sensitive and attenuated phenotype, being inherited from the master donor viruses. The 6:2 reassortant viruses were selected from co-infections between master donor virus and wild type viruses that theoretically may yield as many as 256 combinations of gene segments and thus 256 genetically different viruses. As the time to generate and isolate vaccine viruses is limited and because only 6:2 reassortant viruses are allowed as vaccine viruses, screening needs to be both rapid and unambiguous. The screening of the reassortant viruses by RT-PCRs using master donor virus and wild type virus specific primer sets was described to select both influenza A and influenza B 6:2 reassortant viruses to be used in seasonal and pandemic live attenuated vaccine.

Equine influenza is a contagious disease caused by equine influenza virus which belongs to the orthomyxovirus family. Outbreaks of equine influenza cause severe economic loses to the horse industry and consequently horses in competition are required to be regularly vaccinated against equine influenza. Unlike the existing inactivated vaccines, Equilis Prequenza Te is the only one able to induce protection against clinical disease and virus excretion after a primary vaccination course consisting of two vaccine applications 4–6 weeks apart until the recommended time of the third vaccination. In this paper we describe the duration of immunity profile, tested in an experimental setting according to European legislation, of this inactivated equine influenza and tetanus combination vaccine. In addition to influenza antigen, the formulation contains a second generation ISCOM (the so called ISCOMatrix) as an adjuvant. The vaccine aims at the induction of protection from the primary vaccination course until the time of annual revaccination 12 months later, against challenge with a virulent equine influenza strain. The protection against A/equine/Kentucky/95 (H3N8) at the time of annual revaccination was evidenced by a significant reduction of clinical signs of influenza, a significant reduction of virus excretion and a significant reduction of fever. The effect of the annual revaccination on the duration of immunity against influenza and tetanus was also studied by serology. For tetanus, as a consequence of the 24 months duration of immunity, an alternating annual vaccination schedule consisting of Prequenza and Prequenza Te is proposed after the first three doses of Prequenza Te.

Highly pathogenic avian influenza A viruses of the H5N1 subtype continue to circulate in poultry, and zoonotic transmissions are reported frequently. Since a pandemic caused by these highly pathogenic viruses is still feared, there is interest in the development of influenza A/H5N1 virus vaccines that can protect humans against infection, preferably after a single vaccination with a low dose of antigen. Here we describe the induction of humoral and cellular immune responses in ferrets after vaccination with a cell culture-derived whole inactivated influenza A virus vaccine in combination with the novel adjuvant CoVaccine HT. The addition of CoVaccine HT to the influenza A virus vaccine increased antibody responses to homologous and heterologous influenza A/H5N1 viruses and increased virus-specific cell-mediated immune responses. Ferrets vaccinated once with a whole-virus equivalent of 3.8 μg hemagglutinin (HA) and CoVaccine HT were protected against homologous challenge infection with influenza virus A/VN/1194/04. Furthermore, ferrets vaccinated once with the same vaccine/adjuvant combination were partially protected against infection with a heterologous virus derived from clade 2.1 of H5N1 influenza viruses. Thus, the use of the novel adjuvant CoVaccine HT with cell culture-derived inactivated influenza A/H5N1 virus antigen is a promising and dose-sparing vaccine approach warranting further clinical evaluation.

The aim of this study was to assess the influence of structural properties of N,N,N-trimethyl chitosan (TMC) on its adjuvanticity. Therefore, TMCs with varying degrees of quaternization (DQ, 22-86%), O-methylation (DOM, 0-76%) and acetylation (DAc 9-54%) were formulated with whole inactivated influenza virus (WIV). The formulations were characterized physicochemically and evaluated for their immunogenicity in an intranasal (i.n.) vaccination/challenge study in mice. Simple mixing of the TMCs with WIV at a 1:1 (w/w) ratio resulted in comparable positively charged nanoparticles, indicating coating of WIV with TMC. The amount of free TMC in solution was comparable for all TMC-WIV formulations. After i.n. immunization of mice with WIV and TMC-WIV on days 0 and 21, all TMC-WIV formulations induced stronger total IgG, IgG1 and IgG2a/c responses than WIV alone, except WIV formulated with reacetylated TMC with a DAc of 54% and a DQ of 44% (TMC-RA44). No significant differences in antibody titers were observed for TMCs that varied in DQ or DOM, indicating that these structural characteristics play a minor role in their adjuvant properties. TMC with a DQ of 56% (TMC56) formulated with WIV at a ratio of 5:1 (w/w) resulted in significantly lower IgG2a/c:IgG1 ratios compared to TMC56 mixed in ratios of 0.2:1 and 1:1, implying a shift towards a Th2 type immune response. Challenge of vaccinated mice with aerosolized virus demonstrated protection for all TMC-WIV formulations with the exception of TMC-RA44-WIV. In conclusion, formulating WIV with TMCs strongly enhances the immunogenicity and induces protection against viral challenge in mice after i.n. vaccination. The adjuvant properties of TMCs as i.n. adjuvant are strongly decreased by reacetylation of TMC, whereas the DQ and DOM hardly affect the adjuvanticity of TMC.

A meeting was held at NIBSC, UK in July 2007 to discuss the implications of progress in the use of cell culture systems for the manufacture of vaccines against influenza. Issues discussed included the effect of using eggs and different cell types in strain selection, development of seed viruses to be used in production and the nature of the reagents to be used in determining vaccine potency. Future studies to progress the field were reviewed.

Since the refinement of tissue culture techniques for virus isolation and propagation from the mid 1960s onwards, veterinary virology has received much academic and industrial interest, and has now become a major global industry largely centred on vaccine development against economically important virus diseases of food animals. Bio-tech approaches have been widely used for improved vaccines development. While many viral diseases are controlled through vaccination, many still lack safe and efficacious vaccines. Additional challenges faced by academia, industry and governments are likely to come from viruses jumping species and also from the emergence of virulent variants of established viruses due to natural mutations. Also viral ecology is changing as the respective vectors adapt to new habitats as has been shown in the recent incursion by bluetongue virus into Europe. In this paper the current vaccines for livestock, horses and birds are described in a species by species order. The new promising bio-tech approaches using reverse genetics, non-replicating viral vectors, alpha virus vectors and genetic vaccines in conjunction with better adjuvants and better ways of vaccine delivery are discussed as well.

In order to study the influence of antigen composition, spatial organization of antigen and the route of administration, four cell culture-derived, inactivated, nonadjuvanted influenza vaccine formulations, i.e. whole inactivated virus (WIV), split, subunit and virosome vaccines were prepared from a single antigen batch. We directly compared the immunogenicity and efficacy of these vaccine formulations after intramuscular (i.m.) or intranasal (i.n.) administration in mice. Prime and boost vaccination were followed by a potentially lethal homologous aerosol challenge. For all vaccines, the i.m. route induced higher serum humoral immune responses as compared to the i.n. route and protected all mice against challenge at a dose of 5 microg. Upon i.n. immunization only WIV and split vaccines induced detectable IgG titers and partial protection against challenge but only very low HI titers were induced in almost all mice. WIV induced mainly IgG2a/c titers via both routes, whereas split vaccine induced exclusively IgG1 titers via both routes. Subunit and virosome vaccines induced exclusively IgG1 via the i.m. route. Mucosal sIgA levels were only detected after i.n. vaccination with WIV. Furthermore, vaccines containing all viral components (WIV and split vaccine) induced higher serum HI titers and serum antibody titers than subunit and virosome vaccines. The differences in magnitude and quality of immune responses of split and WIV, having the same composition, are likely related to their distinct spatial organization. In conclusion, the direct comparison between WIV, split, subunit and virosomes, shows that the differences in immune responses between these well known influenza vaccines can be explained by both the composition and particulate structure of these vaccine formulations.

In this article we review important established, newly emergent and potential viral diseases of cats, dogs and rabbits. Topics covered include virus epidemiology, disease pathogenesis, existing and prospective immunoprophylaxis against the viruses. For some feline viruses, notably the immunodeficiency virus, leukaemia virus and peritonitis virus, available vaccines are poorly efficacious but there are good prospects for this. A further challenge for the industry is likely to be due to viruses jumping species and the emergence of more virulent variants of established viruses resulting from mutations as has been the case for the canine parvovirus, coronaviruses and feline calicivirus.

Veterinary vaccines currently available in Europe and in other parts of the world are developed by the veterinary pharmaceutical industry. The development of a vaccine for veterinary use is an economic endeavour that takes many years. There are many obstacles along the path to the successful development and launch of a vaccine. The industrial development of a vaccine for veterinary use usually starts after the proof of concept that is based on robust academic research. A vaccine can only be made available to the veterinary community once marketing authorisation has been granted by the veterinary authorities. This review gives a brief description of the regulatory requirements which have to be fulfilled before a vaccine can be admitted to the market. Vaccines have to be produced in a quality controlled environment to guarantee delivery of a product of consistent quality with well defined animal and consumer safety and efficacy characteristics. The regulatory and manufacturing legislative framework in which the development takes place is described, as well as the trend in developments in production systems. Recent developments in bacterial, viral and parasite vaccine research and development are also addressed and the development of novel adjuvants that use the expanding knowledge of immunology and disease pathology are described.

Conventional equine influenza vaccination schedules consist of a primary course of two vaccinations given 4-6 weeks apart followed by a third vaccination (booster) given approximately 5 months later. In between the primary course and the third vaccination, horses are generally considered not to be adequately protected against influenza. This study aimed to investigate whether Thoroughbred foals would benefit from a vaccination schedule in which the third vaccination was given earlier than in conventional vaccination schedules. The vaccines used were an inactivated whole virus equine influenza vaccine and an inactivated whole virus combination vaccine containing equine influenza and equine herpesvirus antigens. Four groups of foals were vaccinated with the two vaccines according to a conventional and an accelerated vaccination schedule in which the third vaccination was given 14 weeks after the first administration. In both groups, the fourth vaccination was given at the normally recommended interval of 26 weeks after the third vaccination for the combination vaccine and 52 weeks after the third vaccination with the influenza only vaccine. The horses were 4-11 months of age and seronegative for influenza. Immunological responses after vaccination were monitored for several months using the single radial haemolysis test. The results indicated that 28 weeks after the first vaccination, antibody levels in horses vaccinated according to the accelerated schedule were not significantly higher than in horses vaccinated according to the conventional schedule. In addition, the total level of antibody production (area under the curve) was not significantly different at that point although antibody titres were slightly higher (but not significantly so) between 16-30 weeks in the accelerated schedule. Between the third and fourth doses, horses vaccinated according to the accelerated schedule had antibodies against influenza below the level required for clinical protection for 39 and 18 weeks for the influenza only and the combination vaccine, respectively, whereas those vaccinated according to the conventional schedule had antibody titres below the level for clinical protection for 9-15 weeks in the corresponding period for both vaccines. Horses vaccinated according to the accelerated schedule with the combination vaccine had lower antibody titres after the fourth vaccination than those vaccinated according to the conventional schedule after the third vaccination, although antibody titres prior to vaccination were similar. For the influenza only vaccine, titres after the accelerated fourth administration were not different to those after the conventional third vaccination. There was no benefit from early booster vaccinations with the vaccines used in this study, so for these vaccines the conventional schedule provided better protection than the selected accelerated alternative. This may contrast with some other vaccine formulations, although a direct comparison using similar protocols has not been made.

Using the area under the curve (AUC) concept as is commonly used in pharmaceutical bioequivalence studies, the bioequivalence of three equine influenza vaccines was demonstrated. A retrospective analysis was performed using this technique on data generated in three trials in which each of the three vaccines had been used. In total, data from 63 pony and horse foals were used. The AUC of the single radial hemolysis (SRH) titres against Influenza A/equi-1/Prague/56 (Pr/56), A/equi-2/Newmarket-1/93, and A/equi-2/Suffolk/89 (Suf/89) were calculated for each horse. It was concluded that calculation of the AUC from four time-points permitted a suitable estimate for vaccine potency. Using pooled data, it appeared that the AUC permitted better evaluation of vaccine potency than simply considering the highest post vaccinal titre (Titremax). In two studies, a minimal value for the AUC was associated with protection against Influenza (H3N8) challenge 50-153 days later.

Live attenuated influenza vaccine (LAIV) offers the promise of inducing a variety of immune responses thereby conferring protection to circulating field strains. LAIVs are based on cold adapted and temperature sensitive phenotypes of master donor viruses (MDVs) containing the surface glycoprotein genes of seasonal influenza strains. Two types of MDV lineages have been described, the Ann Arbor lineages and the A/Leningrad/17 and B/USSR/60 lineages. Here the safety and immunogenicity of a Madin Darby Canine Kidney - cell culture based, intranasal LAW derived from A/Leningrad/17 and B/USSR, was evaluated in healthy influenza non-naive volunteers 18-50 years of age. In a double-blind, randomized, placebo-controlled design, single escalating doses of 1 x 10(5),1 x 10(6), or 1 x 10(7) tissue culture infectious dose 50% (TCID50) of vaccine containing each of the three influenza virus re-assortants recommended by the World Health Organization for the 2008-2009 season were administered intranasally. A statistically significant geometric mean increase in hemagglutination inhibition titer was reached for influenza strain A/H3N2 after immunization with all doses of LAIV. For the A/H1N1 and B strains, the GMI in HI titer did not increase for any of the doses. Virus neutralization antibody titers showed a similar response pattern. A dose-response effect could not be demonstrated for any of the strains, neither for the HI antibody nor for the VN antibody responses. No influenza like symptoms, no nasal congestions, no rhinorrhea, or other influenza related upper respiratory tract symptoms were observed. In addition, no difference in the incidence or nature of adverse events was found between vaccine and placebo treated subjects. Overall, the results indicated that the LAIV for nasal administration is immunogenic (i.e. able to provoke an immune response) and safe both from the perspective of the attenuated virus and the MDCK cell line from which it was derived, and it warrants further development. (C) 2014 Elsevier Ltd. All rights reserved.

Putative baculovirus helicases have been implicated as playing an important role in viral DNA replication and host specificity. The Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) helicase is therefore of interest since the virus only infects the beet army worm. Sequence analysis of the SeMNPV lef5-p39 (mu 46.5-55.1) region, which is collinear with the 39K-lef5 area in Autographa californica MNPV (AcMNPV), revealed an open reading frame (ORF) of 3666 bp potentially encoding a protein with a molecular mass of 143 kDa. This protein had considerable amino acid sequence similarity (58%) to AcMNPV p143, including seven conserved motifs characteristic of helicases. In cultured insect cells, this SeMNPV ORF is expressed from 4 to 12 h postinfection and its major transcript of 4 kb starts 11 to 12 nt upstream of the putative translational initiation site (ATG). To study their possible role in the specificity of baculovirus DNA replication, the putative AcMNPV and SeMNPV helicase genes were tested for their ability to replicate homologous regions (hrs; putative origins of DNA replication) in a transient DNA replication assay in insect cells. All viral cis- and trans-acting factors were provided as plasmids using either Achr2 or Sehr1 as the DNA replication origin. SeMNPV p143 could not substitute for AcMNPV p143 in the transient assays supplemented with either hr. Similar results were obtained when the SeMNPV and AcMNPV ie1 genes were exchanged. None of the essential AcMNPV trans-acting factors could be complemented by SeMNPV infections to support DNA replication of hrs. These data suggest a specific interaction between baculovirus DNA replication factors to form the replisome and/or between the replisome and the origin of DNA replication.

A new, versatile baculovirus vector was developed for the generation of recombinants in the yeast Saccharomyces cerevisiae and for the expression of foreign proteins in both insect larvae and in insect cells. This vector is based on Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) and exploits the 10-kDa protein promoter (p10) for the expression of the foreign gene. The p10 locus was used for the insertion of a yeast-selectable marker system (ARS-URA-URA3) and of a gene for screening and titration of recombinants in insect cells (β-galactosidase). The polyhedron-positive phenotype of this vector is maintained allowing its use in insect larvae, by feeding polyhedra, and in insect cells, by infecting with budded virus. The generation of this baculovirus vector requires a single recombination step in yeast prior to infection of insect cells, but has the advantage over the vector designed previously (Patel et al., A new method for the isolation of recombinant baculovirus, Nucleic Acids Research 20 (1992) 97–104) that these vectors can also be used in insects.

Continued H5N1 virus infection in humans highlights the need for vaccine strategies that provide cross-clade protection against this rapidly evolving virus. We report a comparative evaluation in ferrets of the immunogenicity and cross-protective efficacy of isogenic mammalian cell-grown, live attenuated influenza vaccine (LAIV) and adjuvanted, whole-virus, inactivated influenza vaccine (IIV), produced from a clade 1 H5N1 6:2 reassortant vaccine candidate (caVN1203-Len17rg) based on the cold-adapted A/Leningrad/134/17/57 (H2N2) master donor virus. Two doses of LAIV or IIV provided complete protection against lethal homologous H5N1 virus challenge and a reduction in virus shedding and disease severity after heterologous clade 2.2.1 H5N1 virus challenge and increased virus-specific serum and nasal wash antibody levels. Although both vaccines demonstrated cross-protective efficacy, LAIV induced higher levels of nasal wash IgA and reduction of heterologous virus shedding, compared with IIV. Thus, enhanced respiratory tract antibody responses elicited by LAIV were associated with improved cross-clade protection.

In April 2009 a new influenza A/H1N1 strain, currently named "pandemic (H1N1) influenza 2009" (H1N1v), started the first official pandemic in humans since 1968. Several incursions of this virus in pig herds have also been reported from all over the world. Vaccination of pigs may be an option to reduce exposure of human contacts with infected pigs, thereby preventing cross-species transfer, but also to protect pigs themselves, should this virus cause damage in the pig population. Three swine influenza vaccines, two of them commercially available and one experimental, were therefore tested and compared for their efficacy against an H1N1v challenge. One of the commercial vaccines is based on an American classical H1N1 influenza strain, the other is based on a European avian H1N1 influenza strain. The experimental vaccine is based on reassortant virus NYMC X179A (containing the hemagglutinin (HA) and neuraminidase (NA) genes of A/California/7/2009 (H1N1v) and the internal genes of A/Puerto Rico/8/34 (H1N1)). Excretion of infectious virus was reduced by 0.5-3 log(10) by the commercial vaccines, depending on vaccine and sample type. Both vaccines were able to reduce virus replication especially in the lower respiratory tract, with less pathological lesions in vaccinated and subsequently challenged pigs than in unvaccinated controls. In pigs vaccinated with the experimental vaccine, excretion levels of infectious virus in nasal and oropharyngeal swabs, were at or below 1 log(10)TCID(50) per swab and lasted for only 1 or 2 days. An inactivated vaccine containing the HA and NA of an H1N1v is able to protect pigs from an infection with H1N1v, whereas swine influenza vaccines that are currently available are of limited efficaciousness. Whether vaccination of pigs against H1N1v will become opportune remains to be seen and will depend on future evolution of this strain in the pig population. Close monitoring of the pig population, focussing on presence and evolution of influenza strains on a cross-border level would therefore be advisable.

Equine influenza is a contagious diseases caused by equine influenza viruses which belong to the orthomyxovirus family. Outbreaks of equine influenza cause severe economic loses to the horse industry and consequently competition horses are required to be regularly vaccinated against equine influenza. Currently available inactivated vaccines are only able to induce protection against clinical disease and virus excretion after a primary vaccination course consisting of three vaccine applications at 4-6 and 22-26 weeks apart, respectively. It has been suggested that these vaccines induce no adequate protection in horses at 22-26 weeks (5 months) in the primary vaccination course (immediately prior to the last booster), despite various alternative vaccination regimens proposed. In this paper we describe the efficacy and safety profile, tested in an experimental setting according to European legislation of a novel inactivated equine influenza vaccine formulation (Prequenza). This formulation consists besides influenza antigen, of second generation ISCOM-Matrix as an adjuvant. The vaccine aims at the induction of protection from the onset of immunity, i.e. after the first two vaccine applications, until the first booster given 5 months later, against challenge with a virulent equine influenza strain. The protection against A/equine/Kentucky/95 (H3N8) was evidenced by a reduction of clinical signs of influenza, a reduction of virus excretion and a reduction of fever. The vaccine was shown to be safe in pregnant mares, foals and is used safely since 2 years as a commercial vaccine in Europe.

A candidate influenza H5N1 vaccine based on cell-culture-derived whole inactivated virus and the novel adjuvant CoVaccineHT (TM) was evaluated in vitro and in vivo. To this end, mice were vaccinated with the whole inactivated influenza A/H5N1 virus vaccine with and without CoVaccineHT (TM) and virusspecific antibody and cellular immune responses were assessed. The addition of CoVaccineHT (TM) increased virus specific primary and secondary antibody responses against the homologous and an antigenically distinct heterologous influenza A/H5N1 strain. The superior antibody responses induced with the CoVaccineHT (TM)-adjuvanted vaccine correlated with the magnitude of the virus-specific CD4+ T helper cell responses. CoVaccineHT (TM) did not have an effect on the magnitude of the CD8+ T cell response. In vitro, CoVaccineHT (TM) upregulated the expression of co-stimulatory molecules both on mouse and human dendritic cells and induced the secretion of pro-inflammatory cytokines TNF-alpha, IL-6, IL-1 beta and IL-12p70 in mouse- and IL-6 in human dendritic cells. Inhibition experiments indicated that the effect of CoVaccineHT (TM) is mediated through TLR4 signaling. These data suggest that CoVaccineHT (TM) also will increase the immunogenicity of an influenza A/H5N1 vaccine in humans. (C) 2009 Elsevier Ltd. All rights reserved.

The purpose of this study was the development and physicochemical and immunological characterization of intranasal (i.n.) vaccine formulations of whole inactivated influenza virus (WIV) coated with N,N,N-trimethyl chitosan (TMC).Synthesized TMCs with a degree of quarternization of 15% (TMC15) or 37% (TMC37) were tested in vitro for their ability to decrease the transepithelial resistance (TEER) of an epithelial cell monolayer. TMC15- and TMC37-coated WIV (TMC15-WIV and TMC37-WIV) were characterized by zeta potential measurements, dynamic light scattering, electron microscopy and gel permeation chromatography. Mice were vaccinated i.n. with selected vaccine formulations and immunogenicity was determined by measuring serum hemagglutination inhibition (HI) and serum IgG, IgG1 and IgG2a/c titers. Also a pulse-chase study with TMCs in solution administered i.n. 2 h prior to WIV was performed. Protective efficacy of vaccination was determined by an aerosol virus challenge.TMC37 induced a reversible decrease in TEER, suggesting the opening of tight junctions, whereas TMC15 did not affect TEER. Simple mixing of (negatively charged) WIV with TMC15 or TMC37 resulted in positively charged particles with TMCs being partially bound. Intranasal immunization with TMC37-WIV or TMC15-WIV induced stronger HI, IgG, IgG1 and IgG2a/c titers than WIV alone. TMC37-WIV induced the highest immune responses. Both TMC15-WIV and TMC37-WIV provided protection against challenge, whereas WIV alone was not protective. Intranasal administration of TMC prior to WIV did not result in significant immune responses, indicating that the immunostimulatory effect of TMC is primarily based on improved i.n. delivery of WIV.Coating of WIV with TMC is a simple procedure to improve the delivery and immunogenicity of i.n. administered WIV and may enable effective i.n. vaccination against influenza.

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It has been recommended that modern equine influenza vaccines should contain an A/equi-1 strain and A/equi-2 strains of the American and European-like subtype. We describe here the efficacy of a modern updated inactivated equine influenza-herpesvirus combination vaccine against challenge with a recent American-like isolate of equine influenza (A/equine-2/Kentucky/95 (H3N8). The vaccine contains inactivated Influenza strains A-equine-1/Prague'56, A-equine-2/Newmarket-1/'93 (American lineage) and A-equine-2/ Newmarket-2/93 (Eurasian lineage) and inactivated EHV-1 strain RacH and EHV-4 strain V2252. It is adjuvanted with alhydrogel and an immunostim. Horses were vaccinated at the start of the study and 4 weeks later. Four, six and eight weeks after the first vaccination high anti-influenza antibody titres were found in vaccinated horses, whereas at the start of the study all horses were seronegative. After the challenge, carried out at 8 weeks after the first vaccination, nasal swabs were taken, rectal temperatures were measured and clinical signs were monitored for 14 days. In contrast to unvaccinated control horses, vaccinated animals shed hardly any virus after challenge, and the appearance of clinical signs of influenza such as nasal discharge, coughing and fever were reduced in the vaccinated animals. Based on these observations, it was concluded that the vaccine protected against clinical signs of influenza and, more importantly, against virus excretion induced by an American-like challenge virus strain. In a second experiment the duration of the immunity induced by this vaccine was assessed serologically. Horses were vaccinated at the start of the study and 6 and 32 weeks later. Anti-influenza antibody titres were determined in bloodsamples taken at the first vaccination, and 2, 6, 8, 14, 19, 28, 32, 37, 41, 45 and 58 weeks after the first vaccination. Vaccinated horses had high anti-influenza antibody titres, above the level for clinical protection against influenza, against all strains present in the vaccine until 26 weeks after the third vaccination.

An adjuvanted vaccine containing inactivated equine influenza, herpesvirus antigens, and tetanus toxoid was administered to young seronegative foals of 8 months of age by deep intramuscular injection in the neck (Group A). The first two vaccinations were given 4 weeks apart. The third was administered 6 months later. Another group of foals (Group B) was vaccinated according to the same scheme at the same time with monovalent equine herpes virus (EHV) vaccine (EHV1.4) vaccine. Antibody responses to the equine influenza (single radial haemolysis; SRH) and tetanus (ToBi ELISA) components of the vaccines were examined from first vaccination until 1 year after the third vaccination. The influenza components of the combination vaccine induced high antibody titres at two weeks after the second vaccination whereafter titres declined until the time of the third vaccination. After the third vaccination, the titres rose rapidly again to remain high for at least 1 year. Antibody titres against tetanus peaked only after the third vaccination but remained high enough to offer protective immunity for at least 1 year. Foals vaccinated with monovalent EHV1.4 remained seronegative for influenza and tetanus throughout the study. Four and a half months after the third vaccination of groups A and B, a third group of animals was vaccinated twice with monovalent EHV1.4 vaccine 4 weeks apart (Group C). Two weeks after the administration of the second dose in the later group, all groups (A, B, C and an unvaccinated control group D) were challenged with EHV-4. Vaccinated foals (Group A, B, C) showed a clear reduction of clinical symptoms and virus excretion after EHV-4 challenge compared with the unvaccinated control foals. No difference could be demonstrated among the vaccinated groups, suggesting that the combination vaccine protects as well as the monovalent vaccine. In EHV1.4-vaccinated foals both antigenic fractions induced clear protection up to 6 months after vaccination (9). It can therefore be anticipated that the efficacy of the combination vaccine against EHV-1 challenge is similar to the efficacy against EHV-1 induced by EHV1.4 vaccination.

The nucleotide sequence of the DNA genome of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV), a group II NPV, was determined and analysed. The genome contains 135611 bp and has a G+C content of 44 mol%. Computer-assisted analysis revealed 139 ORFs of 150 nucleotides or larger; 103 have homologues in Autographa californica MNPV (AcMNPV) and a further 16 have homologues in other baculoviruses. Twenty ORFs are unique to SeMNPV. Major differences in SeMNPV gene content and arrangement were found compared with the group I NPVs AcMNPV, Bombyx mori (Bm) NPV and Orgyia pseudotsugata (Op) MNPV and the group II NPV Lymantria dispar (Ld) MNPV. Eighty-five ORFs were conserved among all five baculoviruses and are considered as candidate core baculovirus genes. Two putative p26 and odv-e66 homologues were identified in SeMNPV, each of which appeared to have been acquired independently and not by gene duplication. The SeMNPV genome lacks homologues of the major budded virus glycoprotein gene gp64, the immediate-early transactivator ie-2 and bro (baculovirus repeat ORF) genes that are found in AcMNPV, BmNPV, OpMNPV and LdMNPV. Gene parity analysis of baculovirus genomes suggests that SeMNPV and LdMNPV have a recent common ancestor and that they are more distantly related to the group I baculoviruses AcMNPV, BmNPV and OpMNPV. The orientation of the SeMNPV genome is reversed compared with the genomes of AcMNPV, BmNPV, OpMNPV and LdMNPV. However, the gene order in the ‘central’ part of baculovirus genomes is highly conserved and appears to be a key feature in the alignment of baculovirus genomes.

The region upstream of the Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) ubiquitin gene contains four near-identical 68-bp-long palindromic repeats. This region, named Sehr6 and located at map unit (m.u.) 88 of the SeMNPV genome on pSeEcoRI-2.2, showed structural homology to previously identified homologous regions (hrs) in a number of other baculoviruses. Hrs function as enhancers of transcription and as putative origins (oris) of baculovirus DNA replication. Five additional hrs (Sehr1-Sehr5) were identified on the SeMNPV genome by Southern blot hybridization with an 18-bp-long oligonucleotide complementary to a sequence conserved within the arms of the four palindromic repeats of Sehr6. Sehr1-Sehr6 were dispersed on the SeMNPV genome at m.u. 8.0, 30.0, 38.5, 51.0, 77.0 and 88.0, respectively. Sequence analysis of these hrs confirmed the presence of palindromic repeats, highly similar to those found in pSeEcoRI-2.2. The number of palindromes varied from one (Sehr4) to nine (Sehr1) per hr. The Sehrs are all present in non-coding regions of the SeMNPV genome and also contain multiple putative transcription recognition sequences. Plasmids containing either of the Sehrs replicated in an SeMNPV-dependent DNA replication assay. The Sehrs were unable to replicate in an AcMNPV-dependent DNA replication assay. This was in contrast to the previously observed SeMNPV non-hr type ori, which replicated in the presence of both AcMNPV and SeMNPV. These data suggest that the replication of SeMNPV and the role of hrs in this process is highly specific.

The genome of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) was screened for the presence of putative origins of DNA replication (oris). Using a transient DNA replication assay, several fragments were identified that underwent SeMNPV-dependent DNA replication in Spodoptera frugiperda cells (Sf-AE-21). Preliminary sequence data revealed the presence of multiple copies of homologous repeats (hrs). Restriction fragment Xbal-F2 showed a distinct sequence reminiscent of Autographa californica and Orgyla pseudotsugata MNPV (AcMNPV and OpMNPV) non-hr oris. Deletion analysis of this fragment indicated that the essential sequences of this putative non-hr ori mapped within a region of 800 bp. Sequence analysis of this region showed a unique distribution of six different (im)-perfect palindromes, several polyadenylation motifs and the occurrence of multiple direct repeats. No sequence homology or similarities to other reported baculovirus oris were detected. The spatial and modular distribution of these motifs are similar to those of the non-hr oris of AcMNPV and OpMNPV. Comparison of baculovirus non-hr and consensus eukaryotic oris revealed no consensus ori but indicated that each of the non-hrs studied so far is unique. From the structural similarity, however, it was concluded that the SeMNPV Xbal-F2 ori represents a baculovirus non-hr type ori. In addition, evidence is provided that SeMNPV renders more specificity to baculovirus DNA replication than AcMNPV.

The baculovirus Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) has high potential for development as a bio-insecticide for control of the beet armyworm (S. exigua). It is highly infectious for S. exigua larvae and its host range is very narrow. A prerequisite for such application is the possibility of growing this virus in large quantities, e.g. in insect cell lines. It was observed, however, that polyhedra of SeMNPV plaque-purified in SeUCR1 cells did not cause larval mortality or morbidity when fed to S. exigua larvae. As this suggested a genetic alteration in in vitro produced SeMNPV, comparative restriction analysis of in vitro and in vivo produced SeMNPV DNA was performed. The restriction patterns of viral DNa from several different plaques always differed from that of the wild-type in the same way, suggesting that a large, single deletion had occurred in the in vitro produced viral genome. In order to localize this deletion more precisely a detailed physical map of the wild-type SeMNPV genome was constructed, using the restriction endonucleases XbaI, BamHI, BglII, PstI, SstI, HindIII and SpeI. In addition, the entire SeMNPV genome was cloned into a library containing five overlapping cosmids and a plasmid library. About 80 restriction sites were located and the orientation of the map was set according to the location of the polyhedrin and p 10 genes. The approximate size of the viral genome was 134 kbp. Based on this map it could be established that mutant SeMNPV, obtained by passage in cell culture, contained a single deletion of approximately 25 kbp between map units 12·9 and 32·3.

Trivalent live attenuated influenza vaccines whose type A components are based on cold-adapted A/Leningrad/134/17/57 (H2N2) (caLen17) master donor virus (MDV) have been successfully used in Russia for decades to control influenza. The vaccine virus comprises hemagglutinin and neuraminidase genes from the circulating viruses and the remaining six genes from the MDV. The latter confer temperature-sensitive (ts) and attenuated (att) phenotypes. The ts phenotype of the vaccine virus is a critical biological determinant of attenuation of virulence. We developed a plasmid-based reverse genetics system for MDV caLen17 to study the genetic basis of its ts phenotype. Mutations in the polymerase proteins PB1 and PB2 played a crucial role in the ts phenotype of MDV caLen17. In addition, we show that caLen17-specific ts mutations could impart the ts phenotype to the divergent PR8 virus, suggesting the feasibility of transferring the ts phenotype to new viruses of interest for vaccine development.