ABSTRACT
ABSTRACT
The efficacies of an oil adjuvanted-inactivated reverse genetics-derived H5 avian influenza virus (AIV) vaccine and an alphavirus replicon RNA particle (RP) AIV vaccine were evaluated in commercial Leghorn chickens. Challenge utilized A/turkey/MN/12582/2015, an isolate representing the U.S. H5N2 Clade 2.3.4.4 responsible for the 2015 highly pathogenic avian influenza (HPAI) epornitic in commercial poultry the United States. As part of a long-term, 36-week study, chickens were challenged at seven weeks of age after receiving a single vaccination, at 18 weeks of age following a vaccine prime-single boost, and at 36 weeks of age after a prime- double-boost. All vaccine programmes reduced virus oropharyngeal and cloacal shedding and mortality compared to the non-vaccinated control birds; however, chickens receiving at least one administration of the RP vaccine generally had diminished viral shedding especially from the cloacal swabbings. A detectable serum antibody response and protection were observed through 18 weeks post-vaccination. Our data suggest that, in conjunction with a comprehensive eradication, enhanced biosecurity and controlled marketing plan, vaccination programmes of commercial layer chickens with novel RP vaccines may represent an important tool for preventing HPAI-related mortalities and decreasing viral load during a catastrophic influenza outbreak.
RESEARCH HIGHLIGHTS
Immunization of poultry following a vaccination schedule consisting of inactivated and RNA particle vaccines offered significant protection against lethal disease following HPAIV challenge.
Virus shedding was significantly (P < 0.05) reduced in chickens vaccinated with either inactivated and/or recombinant vaccines.
Serum antibody titres were not a reliable indicator of protection.
An inactivated vaccine containing 384 HAU of the homologous antigen was unable to induce complete protection.
Introduction
Avian influenza (AI) virus causes one of the most economically important diseases of poultry worldwide. AI is classified by the world organization for animal health (OIE) into two forms, low pathogenicity (LP) and high pathogenicity (HP), based on virulence in chickens (OIE, 2018). HPAI viruses, cause widespread morbidity and mortality in domestic bird populations. Avian influenza virus (AIV) infections occur in many species of wild aquatic birds and are found worldwide (Olsen et al., 2006).
The most widespread HPAI virus is the goose/Guangdong/1996 lineage H5, which is endemic in areas of Africa and Asia, and which has caused recurring outbreaks in Europe since the autumn of 2016 (www.oie.int). Vaccines are routinely used in areas where HPAI virus is endemic, to reduce production losses and to prevent spread (Swayne et al., 2011). Although the U.S. response focuses on eradication without vaccination, after the 2014–2015 outbreak with a Clade 2.3.4.4. goose/Guangdong/1996 lineage H5, vaccines were stockpiled for potential use in healthy, uninfected poultry.
Numerous elements contribute to successful vaccination: antigenic match, immunogenicity, vaccine quality (e.g. antigenic load), general health of the bird and application programme (Spackman & Pantin-Jackwood, 2014). Few studies have been conducted that evaluate immunity after multiple vaccine, application of different types of vaccines, or beyond 3–5 weeks post-vaccination. Duration of immunity and timing of booster vaccinations is critical because longer-lived poultry such as breeders, table-egg layers, and turkeys are a priority for vaccination. This research was performed to determine the efficacy of vaccination of layer chickens using two vaccines currently held in the U.S. Avian Influenza Vaccine Stockpile, including a novel alphavirus replicon RNA particle (RP) vaccine. The studies helped determine the timing and number of vaccine administrations required for protection as assessed by clinical disease and mortality, the reduction or elimination of viral shedding and the duration of immunity conferred.
Materials and methods
Ethical statement
All animal experimental infections and stocking densities were conducted in accordance with the University of Delaware Institutional Animal Care and Use Committee (UD-IACUC) Handbook For Agricultural Animal Care And Use In Research And Teaching (Handbook) and were approved by the UD-IACUC prior to the commencement of trials (project 18-R). The Handbook complies with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 2010).
Animals
Three-hundred-and-twenty, one-day-old, mixed-sex commercial Leghorn chickens were obtained from the commercial supplier, Hy-Line North America, LLC. (West Des Moines, IA). For grow-out and vaccination, birds were housed in HEPA-filtered isolation units (Allentown Inc., Allentown, NJ). Birds had free access to water and commercial feed; a starter ration until 7 weeks of age, a grower ration until 18 weeks of age and a layer ration for the remainder of the trial. At time of challenge, birds were housed in HEPA-filtered isolation units under BSL3 conditions at the USDA licensed Charles C. Allen Biotechnology Laboratory at the University of Delaware (Newark, DE).
Vaccines
The inactivated vaccine was prepared from infectious allantoic fluid inactivated with 0.1% beta-propiolactone (BPL) using rgA/gyrfalcon/Washington/41088/2014 × PR8 H5N1. This isolate was created by reverse genetics, utilizing the haemagglutinin gene from A/gyrfalcon/Washington/41088/2014 H5N8 clade 2.3.4.4 that was engineered to be low pathogenic and the other seven gene segments were from A/Puerto Rico/8/1934 H1N1, a common laboratory influenza A strain which replicates to high titres in embryonating chicken eggs (provided by Dr. David Suarez, USDA, ARS). The vaccine was prepared to contain 384 haemagglutinating units per dose using a commercial oil-based (water-in-oil) adjuvant, Montanide ISA 70VG (70VG) (mineral oil based) (SEPPIC, Inc., Fairfield, NJ), in accordance with the manufacturer’s instructions. The vaccine was stored at approximately 4°C until administered subcutaneously (SQ).
An RP vaccine was also evaluated. The alphavirus replicon transgene was a codon-optimized version of the same low-pathogenic haemagglutinin derived from A/gyrfalcon/Washington/41088/2014 H5N8 (sequence provided by Dr David Suarez, USDA, ARS). This vaccine has been conditionally licensed as Avian Influenza Vaccine, RNA, USDA Product Code 19O5.D0 (Merck Animal Health, Madison, NJ). Briefly, alphavirus replicon RNA particles based on Venezuelan equine encephalitis virus strain TC-83 were prepared using the “promoterless split helper” production process (Kamrud et al., 2010). The vaccine was formulated at a dosage of 107 functional particles per 0.5 ml dose and provided as a frozen liquid without adjuvant. The vaccine was stored at approximately −80°C until administered SQ according to the manufacturer’s instructions.
Haemagglutination inhibition assay
Sera were tested by the haemagglutination inhibition (HI) assay to evaluate vaccine-induced serum antibody levels. The HI assays were performed as described (Pedersen, 2014) using BPL-inactivated HPAIV strain A/turkey/MN/12582/2015 (H5N2) for the antigen (i.e. the challenge virus). Titres were calculated as the reciprocal of the highest dilution where HI activity was observed. Samples with HI titres less than or equal to 8 were considered negative.
Challenge of immunity to HPAIV
At 1-, 7- and 18-weeks of age, chickens were vaccinated SQ according to Table 1. These ages were selected primarily because other vaccinations are routinely administered to commercial layer chickens around these times, so it would be similar to what would be practical for the industry. Serum samples were collected from all chickens prior to re-vaccination or challenge at 7- (trial 1), 18- (trial 2) and 36-weeks of age (trial 3) and tested for the presence of anti-AIV antibodies by HI. Serum samples were stored at −20°C prior to testing.
Table 1. Vaccination schemes of commercial Leghorn chickens for the three challenge of immunity studies.A
All challenge trials (1, 2 and 3) utilized HPAI virus A/turkey/MN/12582/2015 (H5N2). Each challenged bird received a dose of approximately 106 50% embryo infectious dose (EID50)/0.1 ml via the intrachoanal route. The challenge virus titre was confirmed by back-titration in embryonated eggs (Villegas, 2016). Respiratory and digestive system viral shedding titres were determined by collecting oro-pharyngeal (O/P) and cloacal (CL) swabbings on 2, 4- and 7-days post challenge (DPC). Swabbings were placed into 2.0 ml of sterile brain heart infusion (BHI) broth and then stored at −80°C. The samples were thawed once for RNA extraction. Swab sample processing and the AIV matrix (M) gene rRT-PCR assay were performed as described (Preskenis et al., 2017; Spackman et al., 2002). Chickens were considered infected if the virus was detected from the O/P or CL swab samples at any time point. Clinical signs and mortality were recorded daily through to 10 DPC. Clinical signs of disease were scored and recorded as follows: 0 = normal; 1 = mild to moderate signs; 2 = moderate signs, lethargic or not eating; 3 = moderate to severe respiratory signs, severe lethargy, not eating, and neurologic signs; and 4 = dead. Any bird unable to reach food and water was humanely euthanized using methods approved by the UD-IACUC and counted as mortality for the same day. When determining mean death time (MDT) calculations, chickens that were euthanized due to severe illness were counted as mortalities for that day. Serum was collected from the surviving birds at 10 DPC, before these birds were humanely euthanized.
Statistical analysis
Statistical significance between pre-challenge HI antibody titres were determined by ANOVA using the Tukey’s multiple comparison tests. Statistical significance between post-challenge per cent morbidity and mortality figures were determined by chi-square test. Statistical analyses of HI titres and mortality figures were conducted with using the PH Stat (version 4) program (Pearson Education Inc., London, UK) for Excel. All statistical tests were performed using P < 0.05. Superscript lowercase letters in tables and graphs indicate statistical significance between aged-matched groups. Statistical groups are denoted in tables and graphs by lowercase letters. Groups apply to values in the same table column.
Differences in shedding levels between vaccine treatment groups by the same swab type and day post-inoculation were tested for significance by one-way RM ANOVA, Tukey’s test with SigmaPlot 12.0 (Systat software, San Jose, CA). If the virus was not detected in a sample, it was given the value of 0.1 log10 (i.e. it was treated as below the qRT-PCR test limit of detection). A P value of ≤ 0.05 was considered significant.
Results
Trial 1: Protection of seven-week-old commercial Leghorn chickens, six weeks post-vaccination against HPAIV challenge
Prior to challenge at seven weeks, serum antibody titres from each treatment group were determined by HI and were not found to be significantly different (P < 0.05) (Figure 1). The rates of pre-challenge seroconversion (titre >8) by the vaccines were 42% and 35% for the inactivated and RP vaccine treatments, respectively. Sera from the non-vaccinated birds were negative for AIV-specific antibodies prior to challenge.
Published online:
20 May 2019Figure 1. Pre-challenge serum antibody titres six weeks post-vaccination to non-replicating avian influenza vaccines (inactivated, and RNA particle (RP)), by HI assay using the H5N2 HPAI challenge virus (A/turkey/MN/12582/2015) as antigen (trial 1). Statistical significance between mean titres was determined by ANOVA using the Tukey’s multiple comparison test (P < 0.05). There were no significant differences between any groups. Vertical bars represent standard deviation.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2019/cavp20.v048.i04/03079457.2019.1605148/20190619/images/medium/cavp_a_1605148_f0001_ob.jpg"})
Figure 1. Pre-challenge serum antibody titres six weeks post-vaccination to non-replicating avian influenza vaccines (inactivated, and RNA particle (RP)), by HI assay using the H5N2 HPAI challenge virus (A/turkey/MN/12582/2015) as antigen (trial 1). Statistical significance between mean titres was determined by ANOVA using the Tukey’s multiple comparison test (P < 0.05). There were no significant differences between any groups. Vertical bars represent standard deviation.
Protection was measured as the prevention of morbidity and mortality as well as a decrease in oral and cloacal viral shedding. By 3 DPC, none of the birds in the non-vaccinated group survived challenge (Figure 2). Significant (P < 0.05) differences in per cent morbidity and mortality were observed between all treatment groups (Table 2). Only RP vaccine treatment prevented mortality. Although 56% of the birds receiving the inactivated vaccine died after challenge, the MDT was numerically greater than in the non-vaccinated group, indicating that inactivated vaccine delayed the clinical effects of HPAI challenge. With the exception of the non-vaccinated group, per cent morbidity figures exceeded the per cent mortality in a given treatment group (Table 2).
Published online:
20 May 2019Figure 2. Survival curve of Leghorn chickens vaccinated with inactivated and RNA particle (RP) vaccines after H5N2 HPAI virus (A/turkey/MN/12582/2015) challenge six weeks post-vaccination (trial 1).
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2019/cavp20.v048.i04/03079457.2019.1605148/20190619/images/medium/cavp_a_1605148_f0002_oc.jpg"})
Figure 2. Survival curve of Leghorn chickens vaccinated with inactivated and RNA particle (RP) vaccines after H5N2 HPAI virus (A/turkey/MN/12582/2015) challenge six weeks post-vaccination (trial 1).
Table 2. Morbidity, mortality, mean death times, and viral shedding 2-, 4- and 7-days post-challenge (DPC) of seven-week-old commercial layer chickens with HPAIV strain A/turkey/MN/12582/2015 (H5N2) six weeks post-vaccination with inactivated vaccine or RNA particle (RP) vaccine (trial 1).
Both the inactivated and RP vaccine treatments significantly (P < 0.05) decreased oral and cloacal viral shedding titres at 2 DPC (Figure 3(A,B)). The two vaccine treatments could not be compared to the non-vaccinated treatment group beyond 2 DPC as no birds survived beyond 3 DPC. However, significant (P < 0.05) differences continued to be observed between the oral and cloacal viral shedding titres of birds in the inactivated and RP vaccine treatment groups. The RP vaccine substantially reduced viral shed from the cloaca.
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20 May 2019Figure 3. Viral titres from oral (A, C, E) and cloacal swabs (B, D, F) at 2, 4 and 7 days post-challenge (DPC) with HPAIV strain A/turkey/MN/12582/2015 (H5N2). Birds were vaccinated with either the inactivated (Inact) vaccine or the alphavirus RNA particle (RP) vaccine at 1 week of age and challenged 6 weeks later (trial 1) (A, B); or vaccinated with Inact and/or RP vaccines in a prime/boost programme at 1 and 7 weeks of age, respectively, and challenged 11 weeks later (trial 2) (C, D); or vaccinated with Inact and/or RP vaccines in a prime/boost/boost program at 1, 7 and 18 weeks of age, respectively, and challenged 18 weeks later (trial 3) (E,F). Viral titres are expressed as log10 EID50 per 0.1 ml. Different lowercase letters represent statistical differences (P < 0.05) within a sample time (2, 4, or 7 DPC).
Note: Data for non-vaccinated (non-vax) groups were often not available beyond 2 DPC due to death.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2019/cavp20.v048.i04/03079457.2019.1605148/20190619/images/medium/cavp_a_1605148_f0003_oc.jpg"})
Figure 3. Viral titres from oral (A, C, E) and cloacal swabs (B, D, F) at 2, 4 and 7 days post-challenge (DPC) with HPAIV strain A/turkey/MN/12582/2015 (H5N2). Birds were vaccinated with either the inactivated (Inact) vaccine or the alphavirus RNA particle (RP) vaccine at 1 week of age and challenged 6 weeks later (trial 1) (A, B); or vaccinated with Inact and/or RP vaccines in a prime/boost programme at 1 and 7 weeks of age, respectively, and challenged 11 weeks later (trial 2) (C, D); or vaccinated with Inact and/or RP vaccines in a prime/boost/boost program at 1, 7 and 18 weeks of age, respectively, and challenged 18 weeks later (trial 3) (E,F). Viral titres are expressed as log10 EID50 per 0.1 ml. Different lowercase letters represent statistical differences (P < 0.05) within a sample time (2, 4, or 7 DPC).
Note: Data for non-vaccinated (non-vax) groups were often not available beyond 2 DPC due to death.
Trial 2: Protection of 18-week-old commercial Leghorn chickens, 17-weeks post-primary and 11-weeks post-booster vaccinations against HPAIV challenge
Seventeen weeks and 11 weeks post-primary and booster vaccination, respectively, serum antibody titres were evaluated prior to challenge at 18 weeks of age. HI titres of chickens receiving the RP vaccine as the primary or booster vaccine were significantly greater (P < 0.05) than chickens given two inactivated vaccines (Figure 4). Similarly, seroconversion rates (titre >8) of 91% were observed in treatment groups receiving at least one immunization with the RP vaccine. Chickens receiving the inactivated vaccine for both the primary and booster immunizations had the lowest seroconversion rate of any vaccinated group, 34%. Moreover, the HI geometric mean titre (GMT) of the group was not significantly different (P < 0.05) from the non-vaccinated birds. Sera from the non-vaccinated birds were negative for AIV-specific antibodies. By 4 DPC, none of the birds in the non-vaccinated group survived challenge (Figure 5; Table 3). Per cent mortality of the non-vaccinated birds was significantly greater (P < 0.05) than all vaccine treatment groups (Table 3). However, only the RP/RP treatment offered total protection from mortality. All vaccination treatments significantly (P < 0.05) diminished morbidity compared to the non-vaccinated group. Birds receiving the RP/inactivated vaccine protocol displayed numerically lower morbidity than the other treatment groups. Only birds displaying signs of clinical disease died.
Published online:
20 May 2019Figure 4. Pre-challenge serum antibody titres (17 weeks post-primary vaccination /11 weeks post-booster vaccination) from non-replicating avian influenza vaccines (inactivated and RNA particle (RP)), used alone or in combination, by HI assay using the H5N2 HPAI virus challenge virus (A/turkey/MN/12582/2015) as antigen (trial 2). Statistical significance between mean titres was determined by ANOVA using the Tukey’s multiple comparison test (P < 0.05) and represented by different lowercase letters. Vertical bars represent standard deviation. Outliers represented by X.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2019/cavp20.v048.i04/03079457.2019.1605148/20190619/images/medium/cavp_a_1605148_f0004_oc.jpg"})
Figure 4. Pre-challenge serum antibody titres (17 weeks post-primary vaccination /11 weeks post-booster vaccination) from non-replicating avian influenza vaccines (inactivated and RNA particle (RP)), used alone or in combination, by HI assay using the H5N2 HPAI virus challenge virus (A/turkey/MN/12582/2015) as antigen (trial 2). Statistical significance between mean titres was determined by ANOVA using the Tukey’s multiple comparison test (P < 0.05) and represented by different lowercase letters. Vertical bars represent standard deviation. Outliers represented by X.
Published online:
20 May 2019Figure 5. Survival curve of chickens vaccinated with inactivated and RNA particle (RP) vaccines used alone or in combination (primary vaccination/booster vaccination) (trial 2) after H5N2 HPAI virus (A/turkey/MN/12582/2015) challenge 17 weeks post-primary and 11 weeks post-booster vaccination.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2019/cavp20.v048.i04/03079457.2019.1605148/20190619/images/medium/cavp_a_1605148_f0005_oc.jpg"})
Figure 5. Survival curve of chickens vaccinated with inactivated and RNA particle (RP) vaccines used alone or in combination (primary vaccination/booster vaccination) (trial 2) after H5N2 HPAI virus (A/turkey/MN/12582/2015) challenge 17 weeks post-primary and 11 weeks post-booster vaccination.
Table 3. Morbidity, mortality, mean death times, and viral shedding 2-, 4- and 7-days post-challenge (DPC) of 18-week-old commercial layer chickens challenged with HPAIV strain A/turkey/MN/12582/2015 (H5N2) 17 weeks post-primary vaccination and 11 weeks post-booster vaccination (primary vaccination at 1 week of age/booster vaccination at 8 weeks of age) with inactivated (Inact) and/or RNA particle (RP) vaccines (trial 2).
All vaccine treatments significantly decreased (P < 0.05) oral and cloacal viral shedding at 2 DPC as well as oral viral shedding at 4 DPC (Figure 3(C,D)). The lack of non-vaccinated birds surviving the duration of the experiment limited analysis after 4 DPC. However, significant (P < 0.05) differences continued to be observed between the oral and cloacal viral shedding titres of birds in the inactivated and RP vaccine treatment groups. Similar to the primary vaccination challenge trial (Trial 1), the birds in groups receiving the RP as a primary and/or booster vaccine had significantly (P < 0.05) or numerically lower number of chickens shedding challenge virus compared to the non-RP vaccine treatment group (Table 3).
Trial 3: Protection against HPAIV challenge of 36-week-old commercial Leghorn chickens, 35 weeks post-primary, 29 and 18-weeks post-booster vaccinations
Thirty-five-, 29- and 18-weeks post-primary, first booster, and second booster vaccination, respectively, serum antibody titres were evaluated prior to challenge at 36-weeks of age. Complete (100%) seroconversion (titre >8) of birds was observed only in the RP/RP/RP vaccine treatment. Only HI titres of chickens receiving more than one immunization with the RP vaccine were significantly greater (P < 0.05) from the non-vaccinated birds (Figure 6). By 4 DPC, none of the birds in the non-vaccinated group survived challenge (Figure 7; Table 4). All vaccination treatments significantly (P < 0.05) diminished morbidity compared to the non-vaccinated group. Compared to the other vaccine treatments, a significant decrease (P < 0.05) in the per cent morbidity value was observed in the inactivated/inactivated/inactivated treatment group. A similar trend was observed with mortality where per cent mortality of the non-vaccinated birds was significantly greater (P < 0.05) than all vaccine treatment groups (Table 4). As with morbidity, only birds receiving at least one RP vaccination as a primary or booster vaccination treatment were completely protected from post-challenge mortality. The inactivated/ inactivated /inactivated vaccination programme increased the post-challenge mean death time compared to the non-vaccinated birds.
Published online:
20 May 2019Figure 6. Pre-challenge serum antibody titres 35 weeks post-primary vaccination and 28- and 18-weeks post-booster vaccination (primary vaccination/booster vaccination/booster vaccination) (trial 3) with Inact or RP vaccination, by HI assay using homologous antigen (primary vaccination/booster vaccination). Statistical significance between mean titres was determined by ANOVA using the Tukey’s multiple comparison test (P < 0.05) and represented by different lowercase letters. Vertical bars represent standard deviation. Outliers represented by X.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2019/cavp20.v048.i04/03079457.2019.1605148/20190619/images/medium/cavp_a_1605148_f0006_oc.jpg"})
Figure 6. Pre-challenge serum antibody titres 35 weeks post-primary vaccination and 28- and 18-weeks post-booster vaccination (primary vaccination/booster vaccination/booster vaccination) (trial 3) with Inact or RP vaccination, by HI assay using homologous antigen (primary vaccination/booster vaccination). Statistical significance between mean titres was determined by ANOVA using the Tukey’s multiple comparison test (P < 0.05) and represented by different lowercase letters. Vertical bars represent standard deviation. Outliers represented by X.
Published online:
20 May 2019Figure 7. Survival curve of chickens vaccinated with inactivated and RNA particle (RP) vaccines used alone or in combination (primary/booster/booster vaccination) (trial 3) after H5N2 HPAI virus (A/turkey/MN/12582/2015) challenge 35 weeks post-primary vaccination and 28- and 18-weeks post-booster vaccinations with inactivated or RP vaccines.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2019/cavp20.v048.i04/03079457.2019.1605148/20190619/images/medium/cavp_a_1605148_f0007_oc.jpg"})
Figure 7. Survival curve of chickens vaccinated with inactivated and RNA particle (RP) vaccines used alone or in combination (primary/booster/booster vaccination) (trial 3) after H5N2 HPAI virus (A/turkey/MN/12582/2015) challenge 35 weeks post-primary vaccination and 28- and 18-weeks post-booster vaccinations with inactivated or RP vaccines.
Table 4. Morbidity, mortality, mean death time, and virus shedding 2-, 4- and 7-days post-challenge of 36-week-old commercial layer chickens challenged with HPAIV strain A/turkey/MN/12582/2015 (H5N2) 35 weeks post-primary vaccination and 28- and 18-weeks post-booster vaccinations with inactivated (Inact) or RNA particle (RP) vaccine (trial 3).
As seen in trials 1 and 2, all vaccine treatments significantly decreased (P < 0.05) oral and cloacal viral shedding at 2 DPC (Figure 3(E,F)). On 4 DPC, viral shed titres of birds given at least one RP vaccination in the programme were numerically and in some cases significantly lower than the non-vaccinated controls. The lack of non-vaccinated birds surviving the duration of the experiment limited analysis beyond 4 DPC. However, no significant (P < 0.05) differences were observed in oral and cloacal viral shedding between the vaccine treatments. However, birds receiving two or more doses of the RP vaccine displayed numerically lower shedding compared to the other treatment groups.
Discussion
In the past decade, the use of poultry vaccination as a means of HPAI disease control has increased worldwide. Animal health professionals have vaccinated poultry in the hope of limiting viral spread and production losses. Some countries, including the U.S., have avoided vaccination in part due to the impact on the export of poultry and poultry products (Capua & Marangon, 2003).
To date, the most efficacious AI vaccines have been those requiring individual administration of multiple vaccinations making their use most economical in longer-lived birds, such as breeders and table-egg layers. However, few studies evaluated the efficacy of vaccination schedules that include booster vaccinations and/or include a virulent challenge greater than five weeks post-vaccination. Furthermore, new vaccine platforms shown to be effective in other species, such as the RP vaccine, are being evaluated for use in poultry. Each new vaccine should be compared to the well characterized oil-adjuvanted, inactivated vaccines, which are currently the “Gold Standard” AIV vaccines of poultry (Swayne et al., 2011).
Inactivated vaccines have been used for decades and have been found to be generally efficacious if they are good quality, adequately immunogenic and a good antigenic match to the field strain (Swayne, 2009). The classically produced inactivated vaccine utilized in this study contained a recombinant HP HA gene containing an LP cleavage sequence contained in a construct that is capable of replicating to high titres in laboratory host systems.
Inactivated, oil-adjuvanted vaccines did not provide very good protection (defined as: decreased mortality and reduction in virus shedding compared to non-vaccinated birds) as reported in other studies (Chen & Bu, 2009; Bertran et al., 2017; Santos et al., 2017; Kapczynski et al., 2017b). Only after a primer and two booster vaccinations did this vaccine protect against mortality. Among the reported studies which used a similarly prepared inactivated vaccine (Bertran et al., 2017; Kapczynski et al., 2017a,b; Santos et al., 2017) protection was inconsistent. Better protection was observed compared to this study in most conditions, but in one study poor protection was also observed in turkeys 13 weeks post the last vaccination (Santos et al., 2017). The major difference between this study and the previous studies is that a dose of 384 HAU was used here instead of 512 HAU. Potency studies have not been reported with this isolate, but this suggests that 384 HAU is an inadequate dose and could explain the observance of low HI titres and inconsistent protection.
Interestingly, this study showed the best overall protection with the RP vaccine. Although RNA particle vaccines were first reported years ago (Rayner et al., 2002), only in the past few years have these vaccines been evaluated for use in poultry. The alphavirus replicon RP vaccine used in this study has been conditionally approved for use in the US in swine and poultry. The alphavirus RPs are replication defective particles that are unable to spread beyond the initial infected cell (Vander Veen et al., 2012). In this AIV RP vaccine, the structural genes of the alphavirus were replaced with those of the AIV of interest. The alphavirus-based RP vaccines are advantageous because of their flexibility to change the antigen as needed, they can be produced cost-effectively, they are compatible with NA and NP protein based differentiation of infected from vaccinated animals (DIVA) strategies and, unlike inactivated vaccines, they induce cellular mediated immunity (CMI) in addition to antibody (Rayner et al., 2002). Finally, RP vaccine administration would be easier than oil-adjuvanted vaccines as is it less viscous and also poses less of a hazard to humans if accidentally injected.
Better protection was seen in groups which received at least one RP vaccination versus groups that only received the inactivated vaccine. This may be in part due to the host CMI induced by the RP vaccine (Hubby et al., 2007; Rayner et al., 2002). In addition to protecting chickens from mortality and disease, the RP vaccine was effective at reducing shedding as cloacal virus shedding was almost undetectable in chickens receiving at least one dose of the RP vaccine. Kapczynski et al. (2015) observed similar results after the challenge of RP vaccinated chickens. Contrary to this study, the RP vaccine did not perform as well in turkeys (Kapczynski et al., 2017b) as compared to the inactivated vaccine. This could be because the immune response differs between turkeys and chickens (Hartmann et al., 2015; Kandeil et al., 2017; Perozo et al., 2008).
The detection of serum antibodies is a common way to evaluate the host response to AIV vaccines. Individual humoral AIV antibody responses were determined at six (trial 1), 11 (trial 2) and 18 (trial 3) weeks post-vaccination, a duration longer than most published studies. Here, the RP vaccine induced greater HI antibody titres; however, birds immunized with the inactivated vaccine did seroconvert. Consistent with previous studies (Swayne et al., 2015), HI titres in vaccinated chickens did not directly correlate with protection. The serum antibody titres of all treatment groups at 36 weeks of age (trial 3) were similar to those at 18 weeks of age (trial 2), indicating that if an anamnestic response occurred between the administration of the second and third booster, serum antibodies started to wane within that 18-week span between the third vaccination and final pre-challenge bleeding. Santos et al. (2017) found antibody GMTs had decreased 16 weeks post-vaccinations compared to 6 weeks post-vaccination with a similarly produced inactivated vaccine (i.e. vaccine with the same HA produced with the same adjuvant) and the RP product.
In longer-lived birds, multiple administrations of vaccines are necessary to achieve adequate life-long protection. In this study, the vaccines were administered at approximate ages when layer chickens would routinely be handled and vaccinated for other disease agents, thus minimizing the economic impact of a labour-intensive process. When the RP vaccine was included in a vaccine programme, the scheduled application timing was adequate to protect against mortality as well as reduce viral shedding at the different challenge ages using this high-dose homologous challenge model. Based upon results obtained from studies using an inactivated vaccine containing 512 HAU (Bertran et al., 2017; Santos et al., 2017), a single vaccination may be protective. Further work is required to prove such a postulate as well as to determine whether vaccination can maintain egg production levels in layer and breeder flocks.
In summary, we have demonstrated the efficacy of an RP vaccine to protect against an HPAI virus challenge in the context of simulating the administration of scheduled vaccines for commercial Leghorn chickens. It is important to note that with any poultry vaccine efficacy study, the field generated data rarely meet or exceed laboratory protection figures. Our study used antigenically homologous challenge in birds reared in optimal management conditions, free of concurrent infections. Thus, this must be considered when developing a control strategy. Our findings show that, in addition to coordination of culling, quarantine, enhanced surveillance and biosecurity techniques, vaccination represents an additional tool to limit, if not stop the spread of HPAIV in poultry.
| Trial | 1st vaccination at 1 week of age | 2nd vaccination at 7 weeks of age | 3rd vaccination at 18 weeks of age | Number of chickens/group | Time between last vaccination and challenge (weeks) |
|---|---|---|---|---|---|
| 1 | None | – | – | 25 | 6 |
| RP | – | – | 25 | ||
| Inactivated | – | – | 25 | ||
| 2 | None | None | – | 25 | 11 |
| RP | RP | – | 25 | ||
| RP | Inactivated | – | 25 | ||
| Inactivated | Inactivated | – | 25 | ||
| 3 | None | – | – | 25 | 18 |
| RP | RP | RP | 25 | ||
| RP | RP | Inactivated | 25 | ||
| RP | Inactivated | Inactivated | 26 | ||
| Inactivated | Inactivated | Inactivated | 14 |
ALeghorn chickens were obtained from a commercial hatchery at 1 d of age and placed in HEPA filtered isolators. All vaccinations were delivered parenterally via the subcutaneous route.
Notes: None = non-vaccinated; RP = Alphavirus RNA particle vaccine; Inactivated = inactivated oil emulsion vaccine; – = not performed.
| Treatment | Per cent morbidity (number positive/ total) | Per cent mortality (number dead/ total) | Mean death time (days)A | Number of chickens shedding detectable virus (per cent positive) | |||||
|---|---|---|---|---|---|---|---|---|---|
| 2 DPC | 4 DPC | 7 DPC | |||||||
| O/P swab | Cloacal swab | O/P swab | Cloacal swab | O/P swab | Cloacal swab | ||||
| Not vaccinated | 100a (25/25) | 100a (25/25) | 2.1 (2-3) | 25/25a (100) | 25/25a (100) | NT | NT | NT | NT |
| Inactivated | 60b (15/25) | 56b (14/25) | 4.8 (2-8) | 25/25a (100) | 18/25b (72) | 21/21a (100) | 18/21a (86) | 9/13a (69) | 7/13a (54) |
| RP | 32c (8/25) | 0c (0/25) | NA | 23/25a (92) | 2/25c (8) | 21/25a (84) | 9/25b (36) | 5/25b (20) | 0/25b (0) |
Notes: Statistical groups are denoted with lowercase superscripts. NT = not tested. No birds survived for testing. NA = not applicable. No mortality.
AMean death time (in days) of birds succumbing to challenge (first day post-challenge of observed death - last day post-challenge of observed death).
| TreatmentA | Per cent morbidity (number positive/ total) | Per cent mortality (number dead/ total) | Mean death time (days)B | Number of chickens shedding detectable virus (per cent positive) | |||||
|---|---|---|---|---|---|---|---|---|---|
| 2 DPC | 4 DPC | 7 DPC | |||||||
| O/P swab | Cloacal swab | O/P swab | Cloacal swab | O/P swab | Cloacal swab | ||||
| Not vaccinated | 100a (25/25) | 100a (25/25) | 3.3 (3-4) | 25/25a (100) | 25/25b (100) | 7/7a (100) | 7/7a (100) | NT | NT |
| Inact/ Inact | 32b (8/25) | 12b (3/25) | 8 (8) | 25/25a (100) | 4/25a (16) | 21/25a (84) | 14/25b (56) | 21/25a (84) | 14/25a (56) |
| RP/ Inact | 8b (2/25) | 8b (2/25) | 6 (5–7) | 23/25a (92) | 3/25a (12) | 14/25b (56) | 4/25bc (26) | 6/23b (26) | 0/23b (0) |
| RP/ RP | 20b (5/25) | 0b (0/25) | NA | 10/25b (40) | 1/25a (4) | 9/25b (36) | 4/25bc (32) | 8/25b (32) | 3/25b (12) |
Notes: Statistical groups are denoted with lowercase superscripts. NT = not tested. No birds survived for testing. NA = not applicable. No mortality.
APrimary vaccination/booster vaccination.
BMean death time (in days) of birds succumbing to challenge (first day post-challenge of observed death – last day post-challenge of observed death).
| TreatmentA | Per cent morbidity (number positive/ total) | Per cent mortality (number dead/ total) | Mean death time (days)B | Number of chickens shedding virus (per cent positive) | |||||
|---|---|---|---|---|---|---|---|---|---|
| 2 DPC | 4 DPC | 7 DPC | |||||||
| O/P swab | Cloacal swab | O/P swab | Cloacal swab | O/P swab | Cloacal swab | ||||
| Not vaccinated | 100a (24/24) | 100a (24/24) | 2.9 (2-4) | 24/24a (100) | 24/24a (100) | 3/4a (75) | 4/4a (100) | NT | NT |
| Inact/Inact/ Inact | 57 (8/14) | 50 (7/14) | 5.4 (4-7) | 13/14ab (93) | 3/14bc (21) | 14/14a (100) | 11/14ac (79) | 7/7a (100) | 5/7a (71) |
| RP/ Inact/Inact | 0b (0/26) | 0b (0/26) | NA | 22/26b (85) | 5/26bc (19) | 18/26ace (69) | 0/26bc (0) | 15/26bd (58) | 9/26a (35) |
| RP/ RP/ Inact | 0b (0/25) | 0b (0/25) | NA | 6/25c (24) | 0/25bd (0) | 4/25bd (16) | 1/25bc (4) | 0/25bc (0) | 0/25b (0) |
| RP/RP/ RP | 0b (0/25) | 0b (0/25) | NA | 5/25c (20) | 0/25bd (0) | 6/25bd (24) | 0/25bc (0) | 2/25bc (8) | 1/25b (4) |
Notes: Statistical groups are denoted by lowercase superscripts. NT = not tested. No birds survived for testing. NA = not applicable. No mortality.
APrimary vaccination at 1 week of age/8 week booster vaccination/18 week booster vaccination.
BMean death time (in days) of birds succumbing to challenge (first day post-challenge of observed death – last day post-challenge of observed death).
Acknowledgements
The authors gratefully acknowledge Karen Gouge, Krista Murray, Robert Alphin and Scott A. Lee for technical assistance with this work. Contents are solely the responsibility of the authors and do not necessarily represent the official views of the USDA. Mention of trade names or commercial products in this manuscript is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. USDA is an equal opportunity provider and employee.
Disclosure statement
The authors have no potential conflicts of interest to report.