T cell mediated immunity against influenza H5N1 nucleoprotein, matrix and hemagglutinin derived epitopes in H5N1 survivors and non-H5N1 subjects

Abstract

Background Protection against the influenza virus by a specific antibody is relatively strain specific; meanwhile broader immunity may be conferred by cell-mediated immune response to the conserved epitopes across influenza virus subtypes. A universal broad-spectrum influenza vaccine which confronts not only seasonal influenza virus, but also avian influenza H5N1 virus is promising. Methods This study determined the specific and cross-reactive T cell responses against the highly pathogenic avian influenza A (H5N1) virus in four survivors and 33 non-H5N1 subjects including 10 H3N2 patients and 23 healthy individuals. Ex vivo IFN-γ ELISpot assay using overlapping peptides spanning the entire nucleoprotein (NP), matrix (M) and hemagglutinin (HA) derived from A/Thailand/1(KAN-1)/2004 (H5N1) virus was employed in adjunct with flow cytometry for determining T cell functions. Microneutralization (microNT) assay was performed to determine the status of previous H5N1 virus infection. Results IFN-γ ELISpot assay demonstrated that survivors nos. 1 and 2 had markedly higher T cell responses against H5N1 NP, M and HA epitopes than survivors nos. 3 and 4; and the magnitude of T cell responses against NP were higher than that of M and HA. Durability of the immunoreactivity persisted for as long as four years after disease onset. Upon stimulation by NP in IFN-γ ELISpot assay, 60% of H3N2 patients and 39% of healthy subjects exhibited a cross-reactive T cell response. The higher frequency and magnitude of responses in H3N2 patients may be due to blood collection at the convalescent phase of the patients. In H5N1 survivors, the effector peptide-specific T cells generated from bulk culture PBMCs by in vitro stimulation displayed a polyfunction by simultaneously producing IFN-γ and TNF-α, together with upregulation of CD107a in recognition of the target cells pulsed with peptide or infected with rVac-NP virus as investigated by flow cytometry. Conclusions This study provides an insight into the better understanding on the homosubtypic and heterosubtypic T cell-mediated immune responses in H5N1 survivors and non-H5N1 subjects. NP is an immunodominant target of cross-recognition owing to its high conservancy. Therefore, the development of vaccine targeting the conserved NP may be a novel strategy for influenza vaccine design.

Genetic evolution of hemagglutinin and neuraminidase genes of H5N1 highly pathogenic avian influenza viruses in Thailand

“…HA and NA genes, which encode major viral surface glycoproteins, are hypervariable and continuously evolve ( Gutiérrez et al, 2009 ). The biological properties and immune responses against HA and NA glycoproteins of these H5N1 viruses have been previously characterized ( Panaampon et al, 2012 ; Noisumdaeng et al, 2013 ; Noisumdaeng et al, 2014 ; Changsom et al, 2016 ; Noisumdaeng et al, 2021 ). This study comprehensively analyzed the genetic evolution of Thai H5N1 HA and NA nucleotide sequences available in the GenBank database from 2003 to 2010.…”

The role of cell-mediated immunity against influenza and its implications for vaccine evaluation

“…Therefore, membrane expression of this protein is a marker for cytotoxic degranulation of cells and it has been demonstrated that this expression (as measured by flow cytometry) is directly correlated to increased cytotoxicity (as measured by a fluorolysometric assay) ( 110 , 111 ). This method is increasingly being used in influenza infection and vaccine research ( 112 – 115 ). Other methods are the detection of perforins and granzyme B by flow cytometry (but also by ELISA or ELISpot) which are also indirect markers for cytotoxicity ( 85 , 116 , 117 ).…”

Characterization of an intracellular humanized single-chain antibody to matrix protein (M1) of H5N1 virus

“…According to the antigenic characteristics of the virus and its genetic characteristics, influenza viruses are divided into three types: A, B, and C. Influenza A viruses are divided into many subtypes according to different antigens of hemagglutinin HA and neuraminidase NA. HA can be divided into 16 subtypes (H1-H16), and NA has 9 subtypes (N1-N9) [ 4 ]. The combination of different subtypes of HA and NA is responsible for the significant diversity of avian influenza viruses.…”