Immunogenicity and Cross-Protective Efficacy Induced by an Inactivated Recombinant Avian Influenza A/H5N1 (Clade 2.3.4.4b) Vaccine against Co-Circulating Influenza A/H5Nx Viruses.

Controlling avian influenza viruses (AIVs) is mainly based on culling of the infected bird flocks or via the implementation of inactivated vaccines in countries where AIVs are considered to be endemic. Over the last decade, several avian influenza virus subtypes, including highly pathogenic avian influenza (HPAI) H5N1 clade 2.2.1.2, H5N8 clade 2.3.4.4b and the recent H5N1 clade 2.3.4.4b, have been reported among poultry populations in Egypt. This demanded the utilization of a nationwide routine vaccination program in the poultry sector. Antigenic differences between available avian influenza vaccines and the currently circulating H5Nx strains were reported, calling for an updated vaccine for homogenous strains. In this study, three H5Nx vaccines were generated by utilizing the reverse genetic system: rgH5N1_2.3.4.4, rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2. Further, the immunogenicity and the cross-reactivity of the generated inactivated vaccines were assessed in the chicken model against a panel of homologous and heterologous H5Nx HPAIVs. Interestingly, the rgH5N1_2.3.4.4 induced high immunogenicity in specific-pathogen-free (SPF) chicken and could efficiently protect immunized chickens against challenge infection with HPAIV H5N1_2.3.4.4, H5N8_2.3.4.4 and H5N1_2.2.1.2. In parallel, the rgH5N1_2.2.1.2 could partially protect SPF chickens against infection with HPAIV H5N1_2.3.4.4 and H5N8_2.3.4.4. Conversely, the raised antibodies to rgH5N1_2.3.4.4 could provide full protection against HPAIV H5N1_2.3.4.4 and HPAIV H5N8_2.3.4.4, and partial protection (60%) against HPAIV H5N1_2.2.1.2. Compared to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2 vaccines, chickens vaccinated with rgH5N1_2.3.4.4 showed lower viral shedding following challenge infection with the predefined HPAIVs. These data emphasize the superior immunogenicity and cross-protective efficacy of the rgH5N1_2.3.4.4 in comparison to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2.

Efficacy of some avian influenza H5 vaccines against local highly pathogenic avian influenza viruses subtype H5N8 isolated in 2018 and 2020 in Egypt

Commercial inactivated avian influenza H5 vaccine is used as an essential control strategy for avian influenza disease in Egypt. Since the initial outbreaks of highly pathogenic avian influenza H5N8, the virus has diverged with new genotypes and variant viruses continuing to emerge which mainly stand behind vaccination failure. In the present work, four different commercial avian influenza vaccines were inoculated in specific pathogenic free chickens for assessing its efficacy against local highly pathogenic avian influenza H5N8 virus isolated in 2018 and 2020. Two hundred and forty specific pathogenic free chickens were clustered into four groups; each group was inoculated with the corresponding vaccine (60 specific pathogenic free chickens/vaccine). Sixty specific pathogenic free chicks were kept as control unvaccinated group. Sera collected from vaccinated chicken groups at 3rd and 4th week post vaccination were examined for calculating neutralizing antibodies using heterologous highly pathogenic avian influenza H5N8 2018 and 2020. At 4th week post vaccination, vaccinated chickens were challenged; moreover, oropharyngeal swabs were collected from challenged vaccinated chickens to calculate the viral shedding. Our findings revealed the groups vaccinated with vaccine code no 1 and 2 that contains two vaccine strains (H5N1 and H5N8) of local origin exhibited the highest hemagglutination inhibition titer, protection (percent) and reduction in viral shedding titer when examined by highly pathogenic avian influenza H5N8 2018 while, vaccine code no 3 induced lower antibody response, protection (percent) and reduction in viral shedding, but still within satisfactory level when compared to previous groups. When highly pathogenic avian influenza H5N8 2020 was used, it was found the seroconversion rate, protection (percent) and mean titer of reduction of viral shedding decreased in comparison to those recorded for highly pathogenic avian influenza H5N8 2018. Vaccine code no 4 was impotent to either highly pathogenic avian influenza 2018 or 2020. Accordingly, it was recommended to update vaccine strain according to epidemiological condition and used the predominant circulating strain isolate in challenge test(AU)

La vacuna comercial inactivada H5 se utiliza como estrategia esencial de control de la enfermedad de la gripe aviar en Egipto. Desde los brotes iniciales de la gripe aviar altamente patógena H5N8, el virus ha variado al aparecer continuamente nuevos genotipos y variantes virales, que son los principales responsables del fracaso de la vacunación. En el presente trabajo, cuatro vacunas comerciales diferentes contra la gripe aviar se inocularon en pollos libres de patógenos específicos para evaluar su eficacia contra cepas del virus local de la gripe aviar altamente patógeno H5N8 aisladas en 2018 y 2020. Se agruparon 240 pollos pollos libres de patógenos específicos en cuatro grupos, cada uno fue inoculado con la vacuna correspondiente (60 pollos pollos libres de patógenos específicos/vacuna). Sesenta pollos SPF se mantuvieron como grupo control sin vacunar. Los sueros de los pollos vacunados recogidos en la 3ª y 4ª semana después de la vacunación se examinaron para calcular los anticuerpos neutralizantes contra la gripe aviar heteróloga H5N8 2018 y 2020. En la cuarta semana después de la vacunación, los pollos vacunados fueron retados; además, se recogieron hisopados orofaríngeos de los pollos vacunados retados para calcular la diseminación viral. Nuestros resultados revelaron que los grupos vacunados con las vacunas con códigos nº 1 y 2, que contienen dos cepas vacunales (H5N1 y H5N8) de origen local, mostraron el mayor título de inhibición de la hemaglutinación, protección (por ciento) y reducción del título de excreción viral cuando se evaluaron contra la gripe aviar altamente patógena H5N8 2018, mientras que la vacuna con código nº 3 indujo menor respuesta de anticuerpos, protección (por ciento) y reducción de la excreción viral, pero todavía dentro de un nivel satisfactorio en comparación con los grupos anteriores. Al utilizar la vacuna contra la gripe aviar altamente patógena H5N8 2020, se observó que la tasa de seronconversión, la protección (por ciento) y el título medio de reducción de la excreción viral disminuyeron en comparación con los registrados para la gripe aviar altamente patógena H5N8 2018. La vacuna con código nº 4 no fue potente para la gripe aviar altamente patógena de 2018 o de 2020. Por consiguiente, se recomendó actualizar la cepa de la vacuna de acuerdo con las condiciones epidemiológicas y utilizar el aislamiento de la cepa circulante predominante en la prueba de reto(AU)

Alternative method using Real Time PCR for evaluation of inactivated Newcastle disease viral vaccine

The present work aims to establish a new alternative protocol to evaluate in vitro potency of inactivated Newcastle disease virus vaccine using Real Time PCR. Aqueous phases of seven inactivated Newcastle disease virus vaccines batches of different manufacturers were extracted by isopropyl myristate. The Newcastle disease virus antigen of each vaccine sample was determined by a standard Real Time PCR assay. Vaccines were inoculated into separate groups of 3-week-old specific pathogen free chickens using the recommended dose of vaccine. The immunogenicity was assessed for each vaccine by the Newcastle disease virus hemagglutination inhibition antibody titers. Individual serum samples were collected 4 weeks post vaccination, then vaccine efficacy and protection rates were recorded after challenge test of birds vaccinated with the virulent Newcastle disease virus. There is the possibility of using the Real Time PCR as an in vitro assay for vaccine evaluation. The Cycle Threshold values were ranged between 21.17 and 25.23. On the other hand, the hemagglutination inhibition titers ranged between 7.1 log2 to 6.2. The comparison between the Cycle Threshold values of the antigen extracts and the corresponding results of challenge test and in vivo hemagglutination inhibition assays using sera of vaccinated birds proved a strong correspondence between the in vitro and in vivo results(AU)

El presente trabajo pretende establecer un nuevo protocolo alternativo para la evaluación in vitro de la potencia de la vacuna de virus inactivado contra la enfermedad de Newcastle mediante PCR en tiempo real. Las fases acuosas de siete lotes de vacunas inactivadas contra el virus de la enfermedad de Newcastle de distintos fabricantes se extrajeron mediante miristato de isopropilo. El antígeno del virus de la enfermedad de Newcastle de cada muestra de vacuna se determinó mediante un ensayo estándar de PCR en tiempo real. Las vacunas se inocularon en grupos separados de pollos libres de patógenos específicos de 3 semanas de edad utilizando la dosis recomendada de vacuna. La inmunogenicidad se evaluó para cada vacuna mediante los títulos de anticuerpos de inhibición de la hemaglutinación del virus de la enfermedad de Newcastle. Se recogieron muestras individuales de suero 4 semanas después de la vacunación y, a continuación, se registraron la eficacia de la vacuna y los índices de protección tras la prueba de reto de las aves vacunadas con el virus virulento de la enfermedad de Newcastle. Existe la posibilidad de utilizar la PCR en tiempo real como ensayo in vitro para la evaluación de vacunas. Los valores del umbral de ciclo oscilaron entre 21,17 y 25,23. Por otra parte, los títulos de anticuerpos inhibidores de la hemaglutinación oscilaron entre 7,1 log2 y 6,2. La comparación entre los valores del umbral de ciclo de los extractos de antígeno con los resultados correspondientes de la prueba de reto y los ensayos de inhibición de la hemaglutinación in vivo, utilizando sueros de aves vacunadas, demostró una fuerte correspondencia entre los resultados in vitro e in vivo(AU)

Assessment of mycoplasma vaccine efficacy in reducing infection with Newcastle disease virus

The present work recorded the impact of using Mycoplasma gallisepticum vaccines on post-vaccinal response and protection against challenge with Newcastle disease virus. Specific pathogen-free chickens were divided into eight groups of forty chickens each. Group G1 was vaccinated with Mycoplasma gallisepticum live attenuated and Mycoplasma gallisepticum inactivated vaccines. Group G2 was vaccinated with Mycoplasma gallisepticum live attenuated, Mycoplasma gallisepticum inactivated and Newcastle disease inactivated vaccines. Group G3 was vaccinated with Mycoplasma gallisepticum live attenuated vaccine. Group G4 was vaccinated with Mycoplasma gallisepticum live attenuated and Newcastle disease inactivated vaccines. Group G5 was vaccinated with Mycoplasma gallisepticum inactivated vaccine. Group G6 was vaccinated with Mycoplasma gallisepticum inactivated and Newcastle disease inactivated vaccines. Group G7 was vaccinated with Newcastle disease inactivated vaccine. Group G8 was kept as non-vaccinated control. The Newcastle disease hemagglutination inhibition antibodies and mortality percentages were measured. Group G7 recorded the best protective Newcastle disease hemagglutination inhibition antibody titer (7 log2). Group G2 recorded a marginal satisfactory antibody titer (6 log2) after vaccination by the three tested vaccines. The remaining groups revealed unsatisfactory titers ranged from 0-5. The protection levels for G2, G4, G6 and G7 ranged from 70percent to 100percent, but only G2 and G7 were considered protected. G1, G3, G5 and G8 showed typical clinical signs of Newcastle disease. The Mycoplasma gallisepticum vaccines couldn't improve the response to Newcastle disease inactivated vaccine. The results suggest that Mycoplasma gallisepticum vaccination is immunosuppressive rather than immunomodulatory in Newcastle disease vaccination(AU)

En el presente trabajo se registró el impacto de la utilización de vacunas contra Mycoplasma gallisepticum sobre la respuesta posvacunal y la protección frente al reto con el virus de la enfermedad de Newcastle. Pollos libres de patógenos específicos se distribuyeron en ocho grupos de cuarenta pollos cada uno. El grupo G1 se vacunó con vacunas vivas atenuadas e inactivadas contra Mycoplasma gallisepticum. Al grupo G2 se le aplicaron las vacunas: viva atenuada contra Mycoplasma gallisepticum, inactivada contra Mycoplasma gallisepticum e inactivada contra la enfermedad de Newcastle. El grupo G3 se inmunizó con la vacuna viva atenuada contra Mycoplasma gallisepticum; el G4, con las vivas atenuadas contra Mycoplasma gallisepticum e inactivada contra la enfermedad de Newcastle; el G5, con la vacuna inactivada contra Mycoplasma gallisepticum; el G6 con las vacunas inactivadas contra Mycoplasma gallisepticum y la enfermedad de Newcastle; el G7, con la vacuna inactivada contra la enfermedad de Newcastle y el G8 se mantuvo como control no vacunado. Se midieron los anticuerpos de inhibición de la hemaglutinación contra el virus de la enfermedad de Newcastle y los porcentajes de mortalidad. El grupo G7 registró el mejor título de anticuerpos inhibidores de la hemaglutinación contra la enfermedad de Newcastle (7 log2). El grupo G2 registró un título de anticuerpos marginalmente satisfactorio (6 log2) tras la vacunación con las tres vacunas ensayadas. Los demás grupos revelaron títulos insatisfactorios que oscilaban entre 0 y 5. Los niveles de protección de los grupos G2, G4, G6 y G7 oscilaron entre el 70 por ciento y el 100 por ciento, pero sólo G2 y G7 se consideraron protegidos. Los grupos G1, G3, G5 y G8 mostraron signos clínicos típicos de la enfermedad de Newcastle. Las vacunas contra Mycoplasma gallisepticum no pudieron mejorar la respuesta a la vacuna inactivada contra la enfermedad de Newcastle. Los resultados revelan que la vacunación con Mycoplasma gallisepticum es más inmunosupresora que inmunomoduladora en la vacunación contra la enfermedad de Newcastle(AU)

Pathogenicity and vaccine efficacy of two virulent infectious laryngotracheitis virus strains in Egypt.

Infectious laryngotracheitis (ILT) is an economically crucial respiratory disease of poultry that affects the industry worldwide. Vaccination is the principal tool in the control of the disease outbreak. In an earlier study, we comprehensively characterized the circulating strains in Egypt and identified both CEO-like and recombinant strains are dominant. Herein, we investigated the pathogenicity of two virulent strains representing the CEO-like (Sharkia_2018) and recombinant strain (Qalubia_2018). Additionally, we evaluated the efficacy of different commercial vaccines (HVT-LT, CEO, and TCO) against the two isolates in terms of the histopathological lesion scores and the viral (gC) gene load. A total of 270 White Leghorn-specific pathogen-free male chicks were divided into nine groups of 30 birds, each housed in separate isolators. Birds were distributed as follows; one group was non-vaccinated, non-challenged, and served as a negative control. Two groups were non-vaccinated and infected with the two isolates of interest and served as a positive control to test the pathogenicity. Six groups were vaccinated and challenged; two groups were vaccinated with vector vaccine at one day old. The other four groups were vaccinated with either the CEO- or TCO- vaccine (two groups each) at four weeks of age. Three weeks after vaccination, birds were infected with the virulent ILTV isolates. The larynx, trachea, and harderian gland samples were taken at 1, 3, and 7 days post-infection for histopathological lesion score and molecular detection. Notably, The recombinant strain was more virulent and pathogenic than CEO-like ILTV strains. Moreover, the TCO vaccine was less immunogenic than the vector and CEO vaccines.

Selenium nanoparticles enhance the efficacy of homologous vaccine against the highly pathogenic avian influenza H5N1 virus in chickens.

A proper vaccination against avian influenza viruses in chicken can significantly reduce the risk of human infection. Egypt has the highest number of recorded humans highly pathogenic avian influenza (HPAI)-H5N1 infections worldwide despite the widespread use of homologous vaccines in poultry. Enhancing H5N1 vaccine efficacy is ultimately required to better control HPAI-H5N1. The aim of this study is to boost chicken immunity by combined with inactivated HPAI-H5N1 with selenium nanoparticles (SeNPs). The chickens groups 1-3 were fed diets supplemented with SeNPs concentrations (0.25, 0.5, and 1 mg/kg) for 3 weeks and then vaccinated (inactivated HPAI-H5N1). while groups 4,5 and 6 were fed with SeNPs free diets and administered with 0.5 ml of the vaccine combined with 0.02, 0.06, and 0.1 mg/dose of SeNPs and then all groups were challenged with homologous virus 3 weeks post-vaccination (WPV). Group 7, 8 were used as control positive and negative respectively. At 4, 5, and 6 WPV, antibody titer was considerably higher in the group fed a meal supplemented with 1 mg SeNPs/kg. In contrast, both methods of SeNPs supplementation significantly increased the Interleukin 2 (IL2), Interleukin 6 (IL6), and Interferon γ (IFNγ) expressions in the blood cells in a dose-dependent manner, with a higher expression observed in the group that was vaccinated with 0.1 mg/dose. After the challenge, all groups that received SeNPs via diet or vaccines dose showed significant reduction in viral shedding and milder inflammation in lung, trachea, spleen, and liver in addition to higher expression of IL2, IL6, and IFNγ, with the highest expression observed in the group that was vaccinated with 0.1 mg/dose compared the plain vaccinated group. The groups of 1 mg SeNPs/kg and combined vaccinated with 0.1 mg/dose showed the best vaccine efficacy. However, the group vaccinated with 0.1 mg/dose showed the earliest reduction in viral shedding. Overall, SeNPs supplementation in the diet and the administration of the vaccine formula with SeNPs could enhance vaccine efficacy and provide better protection against HPAI-H5N1 in chickens by enhancing cellular immunity and reducing inflammation. We recommend using SeNPs as a vaccine combination or feeding with diet to increase the immunity and vaccine efficacy against H5N1.

Trials for preparation and evaluation of a combined inactivated reassorted H5N1 and Escherichia coli O157 vaccine in poultry.

BACKGROUND AND AIM: Avian influenza (AI), which is one of the major respiratory diseases of poultry, and Escherichia coli (E. coli) have caused major economic losses around the world, including in Egypt. Therefore, in this study, we aimed to produce a vaccine from E. coli O157 and AI H5N1 formulated with Montanide ISA70 for the protection of poultry against both diseases. MATERIALS AND METHODS: We divided one hundred 3-week-old chicks into four groups: Group 1 was vaccinated with prepared inactivated AI H5N1formulated with Montanide ISA70, Group 2 was vaccinated with inactivated E. coli formulated with Montanide ISA70, Group 3 was vaccinated with combined E. coli and AI H5N1 formulated with Montanide ISA70, and Group 4 was an unvaccinated control group. We measured the immune response using the HI (hemagglutination inhibition) test, enzyme-linked immunosorbent assay (ELISA), and the challenge test. RESULTS: We found the three vaccines to be safe and sterile during all periods of examination and observation. The HI test showed that Group 1 exhibited specific antibody titers of 2.3 log2, 4.3 log2, 7.5 log2, 7.8 log2, 8 log2, and 8.1 log2 from week 2 to week 7, respectively, post-vaccination. Group 3 exhibited antibody titers of 3.3 log2, 5.8 log2, 7.8 log2, 8 log2, 8.3 log2, and 8.3 log2 from week 2 to week 7, respectively, post-vaccination. The immune response in both groups reached a high titer at week 6. The combined inactivated E. coli and AI H5N1 vaccine generated a higher immune response than the inactivated AI H5N1 vaccine, and a significant difference exists between the two groups. For Groups 2 and 3, the ELISA antibody titer exhibited its lowest value, 1996.5 and 2036.7, respectively, at week 1 post-vaccination; whereas, both groups exhibited the highest titers, 2227.7 (for Group 2) and 2287.3 (for Group 3), in week 3 post-booster. The ELISA for the combined inactivated E. coli and AI H5N1 vaccine had a higher titer than did the inactivated E. coli vaccine, and a significant difference exists between the two groups. Moreover, the protection rate was higher in Group 3, with 100% for E. coli and 90% for the AI H5N1 vaccine. CONCLUSION: Our findings demonstrate that producing a combined vaccine using E. coli and AI H5N1 formulated with Montanide ISA70 is recommended for protection against both diseases.