The preliminary study showed that the TLL baculovirus-expressed H5 vaccine was able to protect ducks from severe disease and mortality following challenge from a dose of 104.3
of H5N1 virus (Vietnam/1203/04), that is highly pathogenic for ducks. All ducks immunised with low-dose or high-dose vaccines administered with water-in-oil adjuvant were protected but efficacy was lower in the ducks immunised with high-antigen dose without adjuvant, underlining the role of adjuvants in inactivated avian influenza vaccines [1
]. Virus reisolation results further indicated that the birds vaccinated with the baculovirus H5 vaccines only excreted virus briefly and oropharyngeal virus shedding was eliminated (for HDA or LDA groups) compared to unvaccinated control birds.
The comparative vaccination study showed that the TLL HDA vaccine, given as a three dose regime and the TT/H5N2 marker vaccine (2 doses) were as effectively as commercial H5N2 avian influenza vaccine (2 doses) in protecting ducks from severe disease and mortality following challenge with a ten-fold higher dose (105.3EID50) of H5N1 virus (Vietnam/1203/04). All three vaccines showed a similar high level of efficacy in preventing virus shedding from oropharynx and cloaca compared with control ducks. This is a considerable advantage for the control of virus transmission from duck to duck or to other poultry species in the field and in reducing the environmental virus load.
The prechallenge serological responses to H5 HA for the ducks vaccinated with inactivated whole H5N2 virus and TT/H5N2 vaccines are similar to those reported previously with inactivated whole virus H5 vaccines [12
] but the prechallenge H5 antibody response to TLL vaccine was poor and low levels of HI antibody was only detected in some ducks given two or three doses of the TLL HDA vaccine. However, despite low serum H5 antibody responses the TLL vaccinated birds remained protected against HPAI challenge. In both the preliminary study and in the comparative study, TLL vaccinated ducks mounted
4-fold rise in H5 antibody titre after challenge. The TT/H5N2 group also showed a >4-fold rise in H5 titre after challenge but the H5N2 group did not, however there were no significant differences in postchallenge titres between TLL, H5N2 and TT/H5N2 vaccine groups. Other studies have reported that despite the absence of detectable HI antibodies (<1
10) in the prechallenge sera of ducks vaccinated with either of the two lowest vaccine doses of a reverse genetics H5N3 vaccine, there was no virus shedding or disease or deaths after challenge with a duck-lethal H5N1 virus [12
In nature, replication of avian influenza viruses can occur in infected ducks without significant serum antibody response, but despite a poor antibody response, ducks were immune and could resist virus reinfection [28
]. Protection in the vaccinated ducks against HPAI, despite the absence of HI antibody titres, may be due to a priming of duck secretory or mucosal immunoglobulins [30
], or cell-mediated immunity [31
]. The lower prechallenge serum antibody response in TLL ducks may be a function of too low an antigen load in the vaccine for use in ducks compared with the dose required for chickens, which has been discussed previously [31
] and recognised by the manufacturer of the inactivated H5N2 vaccine, who advised use of 1.0
mL dose in ducks instead of the standard 0.5
mL dose in chickens. Another consideration with the baculovirus expressed H5 antigen is that possible differences in glycosylation of insect versus avian cells may have contributed to the low H5 HI antibody titres in vaccinated ducks.
The more frequent reisolation of H5N1 virus in challenged ducks from the oropharynx and only sporadic isolation from cloacal swabs after challenge with A/VN/1203/04 H5N1 HPAI is consistent with previous studies where viral titres shed from the trachea of ducks were higher than from the cloaca for Eurasian H5N1 viruses since 2003 and is believed to be related to a shift in replication efficiency for the upper respiratory tract [4
These challenge studies were conducted using a heterologous H5N1 virus strain and it could be expected that these vaccines would remain efficacious when used in geographical areas where different H5N1 virus clades exist. In contrast to human influenza vaccines, vaccines for poultry do not appear to require close antigenic homology with the haemagglutinin protein and remain able to offer broad cross-protection against diverse field viruses. For example, a single recombinant fowlpox-H5 vaccine was able to clinically protect chickens from challenge by nine different HP H5 strains that had between 87.3 and 100% HA protein sequence similarity with the vaccine strain [2
]. In this study, ducks were challenged with a heterologous clade 1 H5N1 virus and resisted disease and death despite only 94-95% HA1 protein sequence similarity between vaccine and virus strains. However, some recent clade 2.3.2 and clade 2.3.4 H5N1 viruses have shown substantial antigenic diversity from the contemporary H5N1 viruses [33
] and the efficacy of the TLL H5 subunit and H5N2 vaccines used in this study would need to be tested specifically against these new viruses before use in control programmes for them.
Ducks vaccinated with the TT/H5N2 vaccine produced TT antibody responses similar to those in ducks vaccinated with TT/H6N2 vaccine in previous studies [25
]. Antibody responses after the initial vaccination with the TT/H5N2 vaccine were detected in more ducks by C-ELISA (3/7) than indirect ELISA (1/7) but after second vaccination (prechallenge) all ducks were TT antibody positive (prechallenge) at similar levels of reactivity by C-ELISA and indirect ELISA. After H5N1 challenge TT antibody responses by indirect ELISA persisted at similar levels to prechallenge in all ducks, but antibody levels measured by C-ELISA were reduced and two ducks were below the test cut-off. This observation was similar to findings with a previous study with an in-house TT/H6N2 vaccine in Muscovy ducks where indirect ELISA TT antibody persisted at high levels from 6 to 19 weeks postvaccination but C-ELISA results on the same serum showed some reduction in TT antibody response [25
]. Therefore, the indirect ELISA would be better for field use to monitor TT antibody if a H5/TT marker vaccination was being used.
While vaccination remains an important disease control tool for avian influenza, experiences in Hong Kong, Italy, USA and elsewhere showed that vaccination should be part of a programme that incorporates use of quality and efficacious vaccines; quarantine, movement restriction, depopulation and disposal of affected flocks; application or enhancement of flock biosecurity in farms and markets; surveillance to monitor vaccine efficacy as well as field virus circulation; and public awareness on disease prevention and control [3
]. Success with vaccination programs for control of H5N1 HPAI has been quite variable in poultry in East and Southeast Asia, owing to difficulties such as: administering a correct dose to individual birds that are freely roaming and hard to catch; providing an adequate coverage at regular intervals and being inclusive of new hatchlings or purchased birds; maintaining cold chain in tropical climates; observing biosecurity in a village setting; and challenges that local manufacturers may face in ensuring antigen yield, inactivation of killed vaccine and formulation with adjuvants. Specific issues relating to vaccine use for control of H5N1 HPAI includes the risk that vaccination may allow ongoing H5N1 transmission from vaccinated but inapparently infected birds and whether vaccine use may promote antigenic drift and lead to H5N1 virus endemnicity [35
]. These concerns especially relate to H5N1 infection in ducks which are more likely to survive virus infection and produce H5 antibody levels of the same order as ducks that are vaccinated with inactivated whole virus H5 vaccines.
The two vaccines evaluated in this study have shown equivalent efficacy to a commercial inactivated whole virus H5N2 vaccines in ducks, but also have some additional features that may enhance their effectiveness for field use in village poultry avian influenza control systems in developing countries. The recombinant H5 baculovirus vaccine has the major advantage that it does not require embryonated chicken eggs or a high biocontainment facility for production. It should also be relatively straightforward to alter the H5 insert in the baculovirus to match an evolving field H5N1 virus strain. This is a subunit vaccine and after 3 doses in this study produced only weak H5 antibody responses but protected ducks from disease and very significantly reduced virus shedding in H5N1 challenged ducks. The only qualifier with this vaccine is that the third vaccine dose at higher antigen load did produce increased H5 antibody response and this vaccine should be used at a higher antigen dose in ducks, which may induce stronger H5 HI antibody responses. Vaccinated and infected ducks developed an anamnestic antibody response to H5 HA after challenge but because it is a HA subunit vaccine, testing for antibody to influenza A proteins like NP, NS1 or M does confirm the presence of active infection and can be used as a DIVA strategy [21
The TT/H5N2 marker vaccine is as efficacious as the commercial H5N2 vaccine and has the advantage of positively identifying vaccinated ducks. Serological surveillance of ducks for vaccination effectiveness and evidence of virus incursion is particularly difficult where accurate farm records or physical identification of vaccinated birds (leg or wing bands) are not available. Ducks vaccinated with this TT/H5N2 marker vaccine could be very effectively monitored for epidemiological purposes by simple ELISA and HI tests for TT antibody and H5 antibody, respectively. For example, if this vaccine was the approved vaccine for the district or region, simply testing a statistically appropriate sample by TT ELISA would determine the effectiveness of the coverage by the approved vaccine; concurrent testing of H5 antibody would monitor the potency of the vaccine in the field and whether the vaccine handling, storage and application was producing expected levels of flock immunity, and facilitate investigation of poor vaccine responses; testing for TT and H5 antibody during investigation of H5N1 outbreaks in vaccinated flocks would confirm that affected birds had, or had not been effectively vaccinated, and if the vaccine was not effective against the new circulating antigenic strains of the virus; and provide objective measures of risk for H5N1 cases in the district or region based on the level of flock immunity to allow prioritization of avian influenza control activities. Additionally, TT/H5N2 vaccinated ducks in this study, that were challenged with H5N1 viruses, showed significant rises in H5 antibody titre that could provide a signal of recent virus infection. Similar rises in H5 antibody titre have been observed in TT/H5N2 vaccinated chickens after challenge with H5N1 HPAI (Dr Deborah Middleton, CSIRO-AAHL, Geelong, personal communication). Field use of this vaccine could establish normal antibody response curves for TT and H5 antibodies in vaccinated, uninfected ducks. Higher than expected H5 antibody titres, indicating an anamnestic response, would then require further virological investigation.
Extensive vaccination programs for control of H5N1 HPAI have been or are currently being conducted in countries like Vietnam, Indonesia, China and Egypt but outbreaks are still occurring in village poultry systems with domestic ducks being implicated in virus persistence. The two novel vaccines evaluated in this study show equivalent efficacy to an existing commercial vaccine in ducks but they offer advantages for surveillance in village poultry systems in the above counties. The baculovirus recombinant H5 vaccine could be further developed and optimally manufactured in standard vaccine facilities in a developing country without the need for large scale chicken embryo culture facilities. The TT marker can be readily incorporated into any inactivated whole virus or subunit H5 vaccine and used in H5 vaccination programs to provide an effective positive marker of vaccination for surveillance of the small scale and village poultry industries. These tools are recommended for consideration as part of vaccination control programs in countries with ongoing problems of H5N1 HPAI control.