Two Novel Adenovirus Vectors Mediated Differential Antibody Responses via Interferon-α and Natural Killer Cells

ABSTRACT Recombinant adenovirus vectors have been widely used in vaccine development. To overcome the preexisting immunity of human adenovirus type 5 (Ad5) in populations, a range of chimpanzee or rare human adenovirus vectors have been generated. However, these novel adenovirus vectors mediate the diverse immune responses in the hosts. In this study, we explored the immune mechanism of differential antibody responses to SARS-CoV-2 S protein in mice immunized by our previously developed two novel simian adenovirus type 23 (Sad23L) and human adenovirus type 49 (Ad49L), and Ad5 vectored COVID-19 vaccines. Sad23L-nCoV-S and Ad5-nCoV-S vaccines induced the low level of interferon-α (IFN-α) and the high level of antigen-specific antibody responses in wild-type and IFN-α/β receptor defective (IFNAR−/−) C57 mice, while Ad49L-nCoV-S vaccine induced the high IFN-α and low antibody responses in C57 mice but the high antibody response in IFNAR−/− mice. In addition, the high antibody response was detected in natural killer (NK) cells-blocked but the low in follicular helper T (TFH) cells -blocked C57 mice immunized with Ad49L-nCoV-S vaccine. These results showed that Ad49L vectored vaccine stimulated IFN-α secretion to activate NK cells, and then reduced the number of TFH cells, generation center (GC) B cells and plasma cells, and subsequently reduced antigen-specific antibody production. The different novel adenovirus vectors could be selected for vaccine development according to the need for either humoral or cellular or both immune protections against a particular disease. IMPORTANCE Novel adenovirus vectors are an important antigen delivery platform for vaccine development. Understanding the immune diversity between different adenoviral vectors is critical to design the proper vaccine against an aim disease. In this study, we described the immune mechanism of Sad23L and Ad49L vectored vaccines for raising the equally high specific T cell response but the different level of specific antibody responses in mice. We found that Ad49L-vectored vaccine initiated the high IFN-α and activated NK cells to inhibit antibody response via downregulating the number of CD4+ TFH cells leading to the decline of GC B cells and plasma cells.

In our previous studies, two novel adenoviral vectors, Sad23L and Ad49L, derived from a simian adenovirus type 23 (Sad23) or a human adenovirus type 49 (Ad49), were constructed and used for development of Zika and novel coronavirus disease 2019 (COVID- 19) vaccines (10)(11)(12). Both Sad23L and Ad49L vectored vaccines raised the strongly specific T cell response, while Sad23L vectored vaccine induced the high but Ad49L vectored vaccine induced the low level of specific antibody responses (11). Previous studies indicated that type I interferon (IFN-I) had an inverse role for production of antigen-specific antibodies from a chimpanzee adenovirus or lymphocytic choriomeningitis virus (LCMV) infection (9,13). The adenovirus serotypes differ in infectivity, cellular receptor, intracellular trafficking route, and genome CpG content, but these factors have not been conclusively shown to be directly responsible for differentiating immunogenicity (14)(15)(16)(17).
In this study, we sought the role of IFN-a on transgene antigen-specific antibody responses induced by these two novel adenovirus vectored COVID-19 vaccines (Sad23L-nCoV-S and Ad49L-nCoV-S) in comparison with Ad5-nCoV-S vaccine and found that the early status of IFN-a response could be a key factor for triggering activation of natural killer (NK) cells, and subsequently inhibiting production of antigen-specific antibody.
Overall, Sad23L and Ad5 vectored vaccines elicit both higher specific antibody and T cell responses, while Ad49L vectored vaccine induced lower antibody but higher Tcell responses in C57 mice.
injected to Ad49L-nCoV-S immunized C57 mice to block T FH and NK cells for 3 days. The data showed that S-BAb and NAb responses were not detected from both T FH and NK blocked C57 mice in weeks 1 and 2 post injection but detected in weeks 3 and 4 closer to those from C57 mice ( Fig. S1A and S1B), while the IFN-g secreting T-cell response was lower in both T FH and NK blocked C57 mice than that in C57 mice (Fig. S1C) (P , 0.001). The results suggested that the function of T FH cells would determine B and T cell responses.

DISCUSSION
In this study, the immune responses were extensively characterized from novel Sad23L and Ad49L vectored COVID-19 vaccines in comparison with Ad5 vectored vaccine in mice. Sads23L-nCoV-S and Ad5-nCoV-S induced the high antigen-specific antibody response, but Ad49L-nCoV-S induced the low specific antibody response, while all these three vectors mediated the high specific T cell responses and varied insignificantly (Fig. 1). This phenomenon was previously seen in chimpanzee adenovirus type 68 (CAd68), human adenovirus type 4 (Ad4), and human adenovirus type 35 (Ad35) vectors (7)(8)(9). The immune mechanism of this difference between Sad23L and Ad49L vectors was explored for relying on IFN-I signaling that the high level of IFN-a was induced by Ad49L-nCoV-S but not Sad23L-nCoV-S and Ad5-nCoV-S in mice (Fig. 2). Furthermore, the role of IFN-a restricting the antibody response was confirmed by comparing IFNAR 2/2 and normal C57 mice after immunizing with Ad49L-nCoV-S, showing that the specific antibody response was significantly increased in IFNAR 2/2 mice, similar to Sad23L-nCoV-S immunized C57 mice (Fig. 3).
IFN-I was demonstrated for reversely correlating to antiviral B cell response (18,19). A previous study suggested that IFN-I induced by human adenovirus type 28 (Ad28) and Ad35 had multiple effects on T cell immunogenicity (20). There is evidence that NK cells regulated B cell responses; in some studies, NK cells inhibited B cells (21)(22)(23). Interestingly, in our study, when anti-AsialoGM1 polyclonal antibodies were used to block NK cells in C57 mice and then immunized with Ad49L-nCoV-S, the high antigenspecific antibody response was obtained even though a high level of IFN-a in serum (Fig. 5B, G, and H), which were similar to those in IFNAR 2/2 mice immunized with Ad49L-nCoV-S ( Fig. 3E and F), suggesting that the activated NK cells would inhibit specific antibody response. There is evidence showing NK cells restrict the total CD4 1 T cell response (24)(25)(26), of which T FH cells play an essential part in GC B cell formation and subsequent antibody production (27,28). In this study, the mode of NK cells affecting the B cell response was further identified by blocking both NK and T FH cells with specific antibodies, showing that Ad49L vector mediated antibody response was disappeared completely in the early 2 weeks of Ad49L-nCoV-S infection (Fig. S1), suggesting T FH cells played an important role in antibody production.
A study described that IFN-I inhibited B cell response by a chimpanzee adenovirus vector decreasing transgene expression (9). Another study indicated that activation of NK cells was dependent on IFN-a produced by exposure to Ad28 or Ad35, but not to Ad5, in which IFN-a induced activation of NK cells leading to the increased monocyte apoptosis and subsequent loss of vector-insert expression (29). A similar study found that NK cells were activated and accumulated in the liver upon adenoviral infection in vivo, leading to the loss of adenoviral genome and transgene expression in the liver (30). However, these studies did not explain the mechanism of IFN-I affecting antigenspecific B cell response.
IFN-I affecting antigen-specific B cell response has been seen in other viruses as well. Delayed and weakened NAb response alongside with T cell exhaustion represents characteristic features of LCMV as well as of human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) infections (31)(32)(33)(34). Previous studies found that IFN-I suppressed LCMV-specific B cell responses by modulating CD8 1 T cell differentiation and guiding inflammatory monocyte assembling at lymphonodus to prevent antiviral B cell responses (35,36). Our data deduced that IFN-a activated NK cells and consequently decreased the numbers of CD4 1 T FH cells and restricted antigen-specific B cell response in Ad49L vectored vaccine immunized mice.
The mechanisms underlying the differential induction of IFN-I by various adenovirus vectors have not been well defined. Recombinant adenovirus vectors can be sensed by cell-surface or endosomal pattern recognition receptors (PRRs) such as the Toll-like receptors, which can trigger the downstream activation and transcription of antiviral genes, including nuclear factor-k-gene binding (NF-k B), mitogen-activated protein kinases (MAPK), and interferon-regulatory factors (IRFs) (37). One study indicated that IFN-a induction correlated with the permissibility of plasmacytoid dendritic cells (pDCs) to CD46, but not coxsackievirus and adenovirus receptor (CAR)-utilizing Ad serotypes (38,39). And the viral DNA itself can play a crucial role in triggering innate immune responses. The engagement of cyclic GMP-AMP synthase (cGAS) triggers a signaling cascade involving the adaptor stimulator of interferon genes (STING) and activation of the TANK binding kinase 1 (TBK1), which initiate the induction of interferon regulatory factor 3 (IRF3)-responsive genes, such as IFN-I (40). The study showed that loss of the cGAS/STING pathway did not affect viral clearance, and cGAS deficiency had a modest influence on the magnitude of the antiviral humoral immune response to adenovirus infections (40). However, it did not compare with serotypes of adenovirus vectors. Further research is required to determine the key factors responsible for the differential induction of IFN-I by various adenovirus vectors.
In conclusion, besides raising an overall high cellular immunity, these two novel adenovirus vectors mediated differential specific antibody responses, of which Sad23L induced the high but Ad49L induced the low specific antibody activity. Ad49L-vectored vaccine initiated the high IFN-a and activated NK cells to inhibit antibody response via downregulating the number of CD4 1 T FH cells leading to the decline of GC B cells and plasma cells, and the subsequent neutralizing antibody production (Fig. S2). Sad23L or Ad49L vectored vaccine could be considered for prime-boost vaccination in combination or privileged for humoral or cellular immunization against a particular disease.

MATERIALS AND METHODS
Mice and adenoviruses. Normal C57 mice as wild-type control were obtained from the Animal Experimental Centre of Southern Medical University, Guangdong, China. IFNAR -/defective C57 mice were purchased from the Institute of Laboratory Animals Science, CAMS & PUMC. All experiments were conducted in compliance with the guidelines for the care and use of laboratory animals and approved by the Southern Medical University (SMU) Animal Care and Use Committee at Nanfang Hospital, SMU, Guangzhou, China.
In vitro dendritic cell culture and IFN-a measurement. DCs were generated from bone marrow of 4to 6-week-old male C57 mice. Briefly, bone marrow cells were harvested from the femurs and tibiae of mice and cultured in the presence of murine GM-CSF (20 ng/mL) and IL-4 (10 ng/mL) (PeproTech, USA). On day 10, DCs were stimulated with 10 9 PFU Ad5-nCoV-S, Ad49L-nCoV-S or Sad23L-nCoV-S for 24 h, in which the culture supernatants were measured for IFN-a by QuantiCyto Mouse IFN-a ELISA kit (Neobioscience, China).
ELISA. The microtiter plates (Corning, USA) were coated overnight with 2 mg/mL SARS-CoV-2 S proteins (Sino Biological, China). Serum samples were 2-fold serially diluted and S-BAb was detected by ELISA. Secondary antibodies were goat anti-mouse IgG-HRP (Beijing Bersee Science and Technology, Co. Ltd., China). Endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance .0.2 and a ratio of signal than cutoff (S/CO) .1. Log 10 endpoint titers were reported.
NK cell cytotoxicity assay. NK cell cytotoxicity was detected by CyQUANT LDH cytotoxicity kit (eBioscience, San Diego, CA, USA). Briefly, single-cell suspension of mouse splenocytes were then incubated with YAC-1 cells (1 Â 10 4 cells/well) at different effector-to-target cell ratios for 2 h at 37°C, and then 50 mL of each sample culture were transferred to a 96-well flat-bottom plate in triplicate to mix with 50 mL of the substrate for 30 min. Finally, 50 mL of the stop solution were added to each sample well. The absorbance at 490 nm and 680 nm was read, respectively, and the cytotoxicity (%) was calculated by using the following formula: %cytotoxicity ¼ compound-treated LDH activity2spontaneous LDH activity ð Þ = maximum LDH activity ð 2spontaneous LDHÞ Â 100: Statistical analyses. S-BAb and NAb titers between different groups were compared using twotailed t test and one-way ANOVA. IFN-g secreting T cell SFCSs, cytokine rates, YAC-1 cell lysis rates and IFN-a levels among groups were compared by two-tailed t test, one-way ANOVA and Mann-Whitney test. The graphs were conducted by GraphPad Prism version 8 (GraphPad Software, La Jolla, CA, USA). All the statistical analyses were computed with SPSS version 21.0 (SPSS Inc., Chicago, IL, USA). Statistically significant differences are shown with asterisks (*, P , 0.05; **, P , 0.01 and ***, P , 0.001).

SUPPLEMENTAL MATERIAL
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