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. 2023 Aug 22;11(9):1397.
doi: 10.3390/vaccines11091397.

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

Sara H Mahmoud  1 , Ahmed A Khalil  2 , Noura M Abo Shama  1 , Marwa F El Sayed  3 , Reem A Soliman  3 , Naglaa M Hagag  4 , Nahed Yehia  4 , Mahmoud M Naguib  4   5 , Abdel-Sattar Arafa  4 , Mohamed A Ali  1 , Mounir M El-Safty  3 , Ahmed Mostafa  1   6
Affiliations

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

Sara H Mahmoud et al. Vaccines (Basel). .

Abstract

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.

Keywords: AIV H5N1; AIV H5N8; clade 2.3.4.4b; highly pathogenic avian influenza; immunogenicity; vaccine.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Phylogenetic analysis of the surface glycoproteins of the selected candidate vaccine strains versus different circulating isolates of their corresponding clades. (a) Phylogenetic tree of the HA of gene segment of representative strains of H5 gene; (b) Phylogenetic tree of the NA gene segment of representative strains (N1 and N8) gene of the selected candidate vaccine strains. Phylogenetic trees were performed using maximum likelihood methodology based on Akaike criterion with 1000 ultrafast bootstrap replicates in IQ-tree software version 1.1.3 [19] after the selection of the best-fitted model (GTR + F + G4 for both HA and NA). Selected candidate vaccine strains are shown in red.
Figure 2
Figure 2
Genetic constellation of the generated candidate vaccine strains against different H5Nx strains. The HA_Multi-BCS: Multibasic Cleavage Site (highly pathogenic) and HA_Mono-BCS: Monobasic Cleavage Site (low pathogenicity). The asterisk symbol (*) denotes the cleavage position.
Figure 3
Figure 3
Study design including vaccination and sampling time points. The SPF chicks were immunized with the formulated vaccines against the control PBS group at 2-weeks old and samples were collected at the second-week post immunization (WPI). The immunized and control chicks were subjected to challenge infections with homologous and heterologous HPAI H5N1 and H5N8 viruses at 4th WPI. The challenged chicks were monitored for 9 days post challenge for mortality and viral shedding.
Figure 4
Figure 4
Immunogenicity of the formulated H5-type vaccine and cross-reactivity against homologous and heterologous HPAI H5-type strains. Sera from immunized chicks using (a) rgH5N1_2.3.4.4; (b) rgH5N8_2.3.4.4; and (c) rgH5N1_2.2.1.2, were tested using HAI against different H5-type strains of clades 2.3.4.4 and 2.2.1.2.
Figure 5
Figure 5
Survival rate of vaccinated and unvaccinated chickens following challenge with homologous and heterologous HPAI H5-type strains. Immunized chickens were challenged intranasally (106 EID50/0.1 mL) with (a) HPAIV_H5N1_2.3.4.4, (b) HPAIV_H5N8_2.3.4.4 and (c) HPAIV_H5N1_2.2.1.2.
Figure 6
Figure 6
Viral shedding of the immunized chickens at 3-, 5- and 7-days post challenge infections (DPI). The immunized chicken with rgH5N1_2.3.4.4 (G1), rgH5N8_2.3.4.4 (G2) and rgH5N1_2.2.1.2 (G3) were subjected to challenge infections with homologous and heterologous HPAIV H5-type viruses; (a) HPAI_H5N1_2.3.4.4; (b) HPAI_H5N8_2.3.4.4 and (c) HPAI_H5N1_2.2.1.2. The significant differences, compared to the infected unvaccinated control “Control” are indicated (** = p < 0.01, *** = p < 0.001 and non-significant = ns) and highlighted in similar color to each corresponding group.
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