Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Oct;16(5):655-665.
doi: 10.18502/ijm.v16i5.16801.

The heterologous expression of novel recombinant protein composed of HN and F moieties of Newcastle disease virus and immunogenicity evaluation in mouse model

Atena Mozafari  1 , Mehregan Rahmani  1 , Yasaman Yasini Nasab  1 , Shahla Shahsavandi  2 , Mahyat Jafari  1 , Ali Hatef Salmanian  1
Affiliations

The heterologous expression of novel recombinant protein composed of HN and F moieties of Newcastle disease virus and immunogenicity evaluation in mouse model

Atena Mozafari et al. Iran J Microbiol. 2024 Oct.

Abstract

Background and objectives: The rapid spread of Newcastle disease (ND), driven by extensive commercial exchange in the poultry industry, necessitates urgent preventive measures. Although effective vaccines against the Newcastle disease virus (NDV) have been used since 1940, recent outbreaks and the limitations of current vaccines highlight the need for improved solutions. Advances in synthetic biology, reverse vaccinology, molecular biology, and recombinant DNA technology over the past 20 years have led to the development of recombinant vaccines, which offer enhanced protection and broader immunogenic coverage against NDV. This study aimed to express the immunogenic domains of Hemagglutinin Neuraminidase (HN) and Fusion (F) glycoproteins, linked to the heat-labile enterotoxin B subunit (LTB) bio-adjuvant, to develop an effective and reliable recombinant vaccine for NDV.

Materials and methods: In this study, the L(HN)2F protein, composed of the LTB bio-adjuvant and the immunogenic regions of the doubled Hemagglutinin Neuraminidase (HN-HN) and Fusion (F) epitope, was expressed in Escherichia coli. Subcutaneous injection was used to evaluate the humoral immune response in mice and the result was compared with B1 vaccine.

Results: The induction of strong humoral immune responses proved the strong immunoreactivity of the recombinant protein.

Conclusion: The IgG elicited by the recombinant proteins was comparable to that of the commercial B1 vaccine against NDV, indicating its potential as a viable candidate for further development and evaluation as a recombinant vaccine against NDV.

Keywords: Fusion (F) glycoprotein; Hemagglutinin neuraminidase; Immune response; LTB bio-adjuvant; Newcastle disease.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest. Atena Mozafari, Mehregan Rahmani, Yasaman Yasini Nasab, Shahla Shahsavandi, Mahyat Jafari and Ali Hatef Salmanian declare that they have no conflicts of interest.

Figures

Fig. 1
Fig. 1
Schematic view of chimeric L(HN)2F protein and gel electrophoresis of the cloning steps. (A) Diagram of chimeric L(HN)2F; the LTB -HN, HN-HN and HN-F connections were created by four, five and four repeats of EAAAK linkers, respectively. The presence of the different fragments in pET28a (+) was confirmed by (B) Verification of Constructs by PCR, lane 1. ltb (L(HN)2F F and LTB R ~ 350 bp), lane 2. hn (HN-HN F and HN R ~ 250 bp), lane 3. hn-hn (HN-HN F and HN-HN R ~ 550 bp), lane 4. f (F F and L(HN)2F R ~ 450 bp) and lane 5. L(hn)2f (L(HN)2F F and L(HN)2F R ~ 1400 bp) and (C) Verification of Constructs by Restriction Enzyme Digestion (BamHI/HindIII). Lane 1. ltb, lane 2. hn, lane 3. hn-hn, lane 4. f, lane 5 negative control (undigested plasmid) and lane 6. L(hn)2f. M: DNA 100 bp Ladder Mix (Thermo).
Fig. 2
Fig. 2
SDS-PAGE and western blotting analysis of rL(HN)2F. (A) SDS-PAGE analysis of purified rL(HN)2F (lane 1), rLTB (lane 2), rHN (lane 3), rHN-HN (lane 4) and rF (lane 5). Western blotting analysis of purified recombinant proteins with (B) commercial anti-His-tag antibody, (C) anti-Newcastle B1 antibodies. (D) Western blotting analysis of purified recombinant rL(HN)2F, rLTB, rHN, rHN-HN and rF by their specific antibodies. M. protein molecular weight marker (KD). C-. Negative control (protein without His-tag).
Fig. 3
Fig. 3
ELISA assays of anti-sera serial dilutions of recombinant proteins. (A) The reaction of rL(HN)2F antigen with anti-L(HN)2F attained from different boosters and control mice. (B) The cross-reactivity of anti-L(HN)2F IgG with different antigens, including rLTB, rHN, rHN-HN, rF and rL(HN)2F antigens. (C) The cross-reactivity of rL(HN)2F antigen with different antibodies, including anti B1 vaccine strain, anti-LTB, anti-HN, anti-HN-HN, anti-F and anti-L(HN)2F antibodies. Sera from mice immunized with PBS+adjuvant was used as negative control (p < 0.05). The error bar is the standard deviation.
Fig. 4
Fig. 4
Diagrams of IgG titration. Diagram of comparing humoral immune responses assessed by ELISA results of sera from mice immunized with rHN and rHN-HN recombinant proteins (B and C groups) after the first, second, and third boosters.
Fig. 5
Fig. 5
Diagrams of IgG cross-reactivity. (A) Characterization and cross-reactivity analyses of sera from B1 vaccine and rL(HN)2F mice immunized versus all purified recombinant proteins by ELISA. Antibodies titration is calculated at AB492 nm and 1:6400 dilution. (B) Analysis of serum antibody response from the immunized mice with rL(HN)2F and B1 commercial vaccine by ELISA at various days post-first immunization. one-way ANOVA test was performed to analyze and compare treat- ment groups. Control mice were injected with PBS and adjuvant (p < 0.05). The error bar is the standard deviation.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Tan SW, Ideris A, Omar AR, Yusoff K, Hair-Bejo M. Sequence and phylogenetic analysis of Newcastle disease virus genotypes isolated in Malaysia between 2004 and 2005. Arch Virol 2010; 155: 63–70. - PubMed
    1. Alsahami AA, Ideris A, Omar A, Ramanoon SZ, Sadiq MB. Isolation, identification and molecular characterization of Newcastle disease viruses in vaccinated chickens from commercial farms in the Sultanate of Oman. Int J Vet Sci Med 2018; 6: 248–252. - PMC - PubMed
    1. Ma F, Zhang E, Li Q, Xu Q, Ou J, Yin H, et al. A Plant-produced recombinant fusion protein-based Newcastle disease subunit vaccine and rapid differential diagnosis platform. Vaccines (Basel) 2020; 8: 122. - PMC - PubMed
    1. Bello MB, Yusoff K, Ideris A, Hair-Bejo M, Jibril AH, Peeters BP, et al. Exploring the prospects of engineered Newcastle disease virus in modern vaccinology. Viruses 2020; 12: 451. - PMC - PubMed
    1. Bello MB, Mahamud SNA, Yusoff K, Ideris A, Hair-Bejo M, Peeters BPH, et al. Development of an effective and stable genotype-matched live attenuated Newcastle disease virus vaccine based on a novel naturally recombinant Malaysian isolate using reverse genetics. Vaccines (Basel) 2020; 8: 270. - PMC - PubMed

LinkOut - more resources