The major mechanism for determination of HCV infection outcomes has not been fully described, particularly in the early phase of the “window-period” of infection. Based on two groups of marmosets infected with HCV-CE1E2p7/GBV-B chimeric virus (HCV chimera) or GBV-B, the immune mechanism correlating with the different outcomes of virus infections was explored in this study. HCV chimera containing the entire HCV core and envelope proteins (CE1E2p7) and GBV-B RNA were intrahepatically injected into four marmosets in each group, respectively. Blood samples were taken from individual animals in an interval of 2 weeks. Viral load and specific T cell responses were detected in two groups of HCV chimera- and GBV-B-infected marmosets. HCV chimera-infected marmosets appeared to have a virally persistent infection over 6 months post inoculation of the virus. Of these, the specific IFN-γ-secretion T cell response slowly developed over 13 to 19 weeks and was maintained at a relatively low level with 40–70 SFC/10⁶ PBMCs, while the specific Treg cell response was rapidly activated over 3 weeks and was maintained at a high level around 5% among lymphocytes. In contrast, GBV-B-infected marmosets presented spontaneous viral clearance within 6 months; the specific IFN-γ-secretion T cell response was quickly established over 5 to 7 weeks and was maintained at a high level with 50–130 SFC/10⁶ PBMCs, while the specific Treg cell response was inactivated and maintained at a baseline below 3% among lymphocytes. In conclusion, the HCV structural proteins inducing immune suppression in the early phase of HCV infection contributed to the viral persistence, of which the activation of Treg cells might play an important role in the inhibition of an effective T cell antiviral response.

Background: Natural killer (NK) cells are key effectors of innate immunity with broad-spectrum anti-tumor activity. However, peripheral blood-derived NK (PBNK) cells are typically quiescent, which limits their therapeutic utility. This study aimed to develop an efficient strategy for the in vitro activation and expansion of PBNK cells and then evaluate their potential anti-tumor efficacy in vitro and vivo. Methods: NK cells were isolated from healthy blood donors’ peripheral blood and stimulated with anti-CD16 and anti-CD137 antibodies in the presence of interleukin-2 (IL-2) and interleukin-15 (IL-15) under serum-free conditions, generating super NK (SNK) cells. The expression levels of activating and inhibitory receptors on the expanded SNK cells were assessed by flow cytometry. Cytotoxicity against tumor cells was assessed at various effector-to-target (E:T) ratios in vitro. In vivo, anti-tumor efficacy was evaluated in K562-engrafted NSG mice. RNA sequencing was performed to identify differentially expressed genes (DEGs) between SNK and PBNK cells. Results: Stimulation with anti-CD16 and anti-CD137 antibodies resulted in significant expansion of donor-derived NK cells, with over 861.9 ± 48.84-fold expansion (n = 5) within 15 days of culture. SNK cells exhibited significantly elevated expression of activating receptors, including NKG2D. Functionally, SNK cells demonstrated superior cytotoxicity compared with PBNK cells across all tested E:T ratios in vitro and higher expressions of the effector molecules interferon-gamma (IFN-γ) and granzyme B (Gzm B). In vivo, adoptive SNK cell transfer resulted in significant tumor suppression and prolonged survival in a dose-dependent manner. Transcriptomic analysis revealed significant enrichment of DEGs associated with cytokine and chemokine signaling, immune activation, and cytotoxic effector function compared with the PBNK cells. Conclusions: Anti-CD16/CD137 antibody stimulation, in combination with IL-2 and IL-15, facilitates robust activation and rapid expansion of functionally enhanced NK cells from peripheral blood. The resulting SNK cells demonstrated enhanced anti-tumor efficacy both in vitro and in vivo and may be used as allogeneic NK cell-based immunotherapy in future cancer treatment strategies.

COVID-19 vaccines are being developed urgently worldwide, among which single-shot adenovirus vectored vaccines represent a major approach. Here, we constructed two novel adenovirus vectored COVID-19 vaccine candidates on simian adenovirus serotype 23 (Sad23L) and human adenovirus serotype 49 vectors (Ad49L) carrying the full-length gene of SARS-CoV-2 spike protein (S), designated Sad23L-nCoV-S and Ad49L-nCoV-S vaccines, respectively. The immunogenicity elicited by these two vaccine strains was individually evaluated in mice. Specific humoral and cellular immune responses were proportionally observed in a dose-dependent manner, and stronger response was obtained by boosting. Furthermore, five rhesus macaques were intramuscularly injected with a dose of 5×10 ⁹ PFU Sad23L-nCoV-S vaccine for prime vaccination, followed by boosting with 5×10 ⁹ PFU of Ad49L-nCoV-S vaccine at 4-week interval. Three macaques were injected with Sad23L-GFP and Ad49L-GFP vectorial viruses as negative controls. Both mice and macaques tolerated well the vaccine inoculations without detectable clinical or pathologic changes. In macaques, prime-boost vaccination regimen induced high titers of 10 ^3.16 S-binding antibody (S-BAb), 10 ^2.75 cell receptor binding domain (RBD)-BAb and 10 ^2.38 neutralizing antibody (NAb) to pseudovirus a week after boosting injection, followed by sustained high levels over 10 weeks of observation. Robust IFN-γ secreting T-cell response (712.6 SFCs/10 ⁶ cells), IL-2 secreting T-cell response (334 SFCs/10 ⁶ cells) and intracellular IFN-γ expressing CD4 ⁺ /CD8 ⁺ T cell response (0.39%/0.55%) to S peptides were detected in the vaccinated macaques. It was concluded that prime-boost immunization with Sad23L-nCoV-S and Ad49L-nCoV-S vaccines can safely elicit strong immunity in animals in preparation of clinical phase 1/2 trials.