Dynamics of Neutralizing Antibodies and Binding Antibodies to Domains of SARS-CoV-2 Spike Protein in COVID-19 Survivors.

Neutralizing antibody level is used to predict immune protection against SARS-CoV-2 infection. Spike protein of SARS-CoV-2 is a major target for virus-neutralizing antibody. A number of neutralizing epitopes were mapped on receptor binding domain (RBD) and N-terminal domain (NTD) of S1 subunit of the spike. Anti-SARS-CoV-2 antibody usually decreases over time after recovery. Level of neutralizing antibody and binding antibody to several domains from COVID-19 recovered patients was observed longitudinally in this study. Sequentially collected serum samples from 35 patients demonstrated both similar and different trends of neutralizing antibodies versus binding antibodies to each domain. Twenty-three individuals showed similarly decreasing pattern of neutralizing titer, binding antibodies to RBD, NTD, fusion protein (S2), and nucleocapsid (NP). Interestingly, eight individuals had stably high neutralizing titer (≥320) for 3-12 months, whereas their binding antibodies to RBD, NTD, and NP rapidly decreased. Moreover, their binding antibodies to S2 were stable over time similar to the persistence of neutralizing antibody levels. The long-lasting antibody to S2 suggested an anamnestic response to cross-reactive epitopes from previous infections with other related coronaviruses. These data indicate a difference in kinetics and longevity of antibodies to various domains and epitopes of the SARS-CoV-2 proteins. A better understanding in this difference may help improve vaccine design to induce long-lasting immunity to COVID-19.

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

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.

Generation and preclinical immunogenicity study of dengue type 2 virus-like particles derived from stably transfected mosquito cells.

Recent phase IIb/III trials of a tetravalent live attenuated vaccine candidate revealed a need for improvement in the stimulation of protective immunity against diseases caused by dengue type 2 virus (DENV-2). Our attempts to develop particulate antigens for possibly supplementing live attenuated virus preparation involve generation and purification of recombinant DENV-2 virus-like particles (VLPs) derived from stably (prM+E)-expressing mosquito cells. Two VLP preparations generated with either negligible or enhanced prM cleavage exhibited different proportions of spherical particles and tubular particles of variable lengths. In BALB/c mice, VLPs were moderately immunogenic, requiring adjuvants for the induction of strong virus neutralizing antibody responses. VLPs with enhanced prM cleavage induced higher levels of neutralizing antibody than those without, but the stimulatory activity of both VLPs was similar in the presence of adjuvants. Comparison of EDIII-binding antibodies in mice following two adjuvanted doses of these VLPs revealed subtle differences in the stimulation of anti-EDIII binding antibodies. In cynomolgus macaques, VLPs with enhanced prM cleavage augmented strongly neutralizing antibody and EDIII-binding antibody responses in live attenuated virus-primed recipients, suggesting that these DENV-2 VLPs may be useful as the boosting antigen in prime-boost immunization. As the levels of neutralizing antibody induced in macaques with the prime-boost immunization were comparable to those infected with wild type virus, this virus-prime VLP-boost regimen may provide an immunization platform in which a need for robust neutralizing antibody response in the protection against DENV-2-associated illnesses could be tested.