Adenoviruses vectored hepatitis C virus vaccine cocktails induce broadly specific immune responses against multi-genotypic HCV in mice.

BACKGROUND: Hepatitis C virus (HCV) vaccines are an urgent need to prevent hepatitis C and its further progression of hepatocellular carcinoma. Since the promising T cell based chimpanzee adenovirus and modified vaccinia virus Ankara vectorial HCV vaccines were failed in clinical phase II trial, the vaccine designs to improve protection efficacy in combination of cellular and humoral immunity have been hypothesized against multi-genotypic HCV. METHODS: Eight HCV vaccine strains were constructed with two novel adenovirus vectors (Sad23L and Ad49L) encoding E1E2 or NS3-5B proteins of HCV genotype (Gt) 1b and 6a isolates, covering 80 % HCV strains prevalent in south China and south-east Asia. Eight HCV vaccine strains were grouped into Sad23L-based vaccine cocktail-1 and Ad49L-based vaccine cocktail-2 for vaccinating mice, respectively. RESULTS: The immunogenicity of a single dose of 107-1010 PFU HCV individual vaccines was evaluated in mice, showing weak specific antibody to E1 and E2 protein but a dose-dependent T cell response to E1E2/NS3-5B peptides, which could be significantly enhanced by boosting with an alternative vector vaccine carrying homologous antigen. Prime-boost vaccinations with vaccine cocktail-1 and cocktail-2 induced significantly higher cross-reactive antibody and stronger T cell responses to HCV Gt-1b/6a. The high frequency of intrasplenic and intrahepatic NS31629-1637 CD8+ T cell responses were identified, in which the high proportion of TRM and TEM cells might play an important role against HCV infection in liver. CONCLUSIONS: Prime-boost regimens with HCV vaccine cocktails elicited the broad cross-reactive antibody and robust T cell responses against multi-genotypic HCV in mice.

A novel simian adenovirus-vectored COVID-19 vaccine elicits effective mucosal and systemic immunity in mice by intranasal and intramuscular vaccination regimens.

IMPORTANCE: The essential goal of vaccination is to generate potent and long-term protection against diseases. Several factors including vaccine vector, delivery route, and boosting regimen influence the outcome of prime-boost immunization approaches. The immunization regimens by constructing a novel simian adenovirus-vectored COVID-19 vaccine and employing combination of intranasal and intramuscular inoculations could elicit mucosal neutralizing antibodies against five mutant strains in the respiratory tract and strong systemic immunity. Immune protection could last for more than 32 weeks. Vectored vaccine construction and immunization regimens have positively impacted respiratory disease prevention.

Characterization of Pre-Existing Neutralizing Antibody to Human Adenovirus Types 5 and 49 and Simian Type 23 in Chinese Population.

Recombinant adenovirus vector has been widely used in vaccine development. Due to the pre-existing immunity of human adenovirus type 5 (HAd5) in humans, a range of rare human and chimpanzee adenovirus vectors have been developed. In the previous study, we constructed novel adenovirus vector Sad23L and Ad49L based on simian adenovirus type 23 (SAd23) and human adenovirus type 49 (HAd49), which were used in the development of ZIKV and COVID-19 vaccines. However, the levels of pre-existing neutralizing antibody (NAb) of HAd49 and SAd23 remain unclear in China. In this study, we measured NAbs titers of HAd5, HAd49, and SAd23 in 600 healthy blood donors from 6 regions across China. NAb titer of HAd49 or SAd23 was significantly lower than that of HAd5 (p < 0.001). There was no significant difference in seroprevalence and NAb titers of three adenoviruses between male and female donors. The seropositive rates of HAd5 and SAd23 increased with age growth in a positive correlation (p < 0.01), while in contrast to HAd5, HAd49, and SAd23 had a low level of pre-existing immunity in Chinese population, which suggested that Ad49L and Sad23L vectors could be used in vaccine development for humans.

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

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/ß 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.

Early Phase of Specific Cellular Immune Status Associates with HCV Infection Outcomes in Marmosets.

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.

Occult Hepatitis B Virus Infection and Liver Fibrosis in Chinese Patients.

BACKGROUND: The impact of hepatitis B surface antigen (HBsAg)-negative/hepatitis B virus (HBV) DNA-positive occult HBV infection (OBI) on the severity of liver fibrosis remains unclear. METHODS: A total of 1772 patients negative for HBsAg but positive for antibody to hepatitis B core antigen (HBcAg), stratified by the presence or absence of OBI, were selected for long-term carriage leading to elevation of ≥2 of 4 liver fibrosis indexes-hyaluronic acid (HA), laminin, type III procollagen peptide (PCIII), and type IV collagen (CIV)-at testing in a Chinese hospital. Patients were tested for serum viral load, HBV markers, and histopathological changes in liver biopsy specimens. RESULTS: OBI was identified in 148 patients with liver fibrosis (8.4%), who had significantly higher levels of HA, laminin, PCIII, and CIV than 1624 fibrotic patients without OBI (P < .05). In 36 patients with OBI who underwent liver biopsy, significant correlations were observed between OBI viral load and serum HA levels (P = .01), PCIII levels (P = .01), and pathological histological activity index (HAI) scores (P < .001), respectively; HAI scores and PCIII levels (P = .04); HBcAg immunohistochemical scores and HA levels (P < .001); and HBcAg immunohistochemical scores and PCIII levels (P = .03). Positive fluorescent in situ hybridization results were significantly more frequent in patients with OBIs (80.6% vs 37.5% in those without OBIs). Among patients with OBIs, HBcAg was detected in the liver tissue in 52.8% and HBsAg in 5.6%. CONCLUSIONS: OBI status appears to be associated with liver fibrosis severity.

Vaccination of cats with Sad23L-nCoV-S vaccine candidate against major variants of SARS-CoV-2.

Cats are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and risk transmitting viruses to naive cats or humans. Here, based on our novel adenovirus-vectored COVID-19 vaccine, the immunogenicity of Sad23L-nCoV-S vaccine was evaluated in cats by prime-boost vaccinations. Five cats were primed with a dose of 108 plaque-forming units (PFUs) Sad23L-nCoV-S vaccine and then boosted with an equal dose of same vaccine at a 4-week interval. Cat serum neutralizing antibody (NAb) titers (the sample dilution at which 50% inhibitory concentration [IC50]) were measured as IC50 15,849 to wild-type strain, IC50 6,591 to Alpha, IC50 2,315 to Beta, IC50 2,744 to Gamma, IC50 1,848 to Delta, and IC50 318 to Omicron variants of pseudotyped SARS-CoV-2 viruses at week 6 post-prime vaccination. All NAb levels to these five variants were ≥IC50 49 from vaccinated cats at week 10, while 48.8% to Delta and 100% to Omicron variants were

Hepatitis B Virus-Specific Cellular Immunity Contributes to the Outcome of Occult Hepatitis B Virus Infection.

There is little known of immunologic factors leading to the occurrence of occult HBV infection (OBI). Specific cellular immune response to hepatitis B virus (HBV) core/pol peptides was compared between blood donor populations, including 37 OBIs, 53 chronic HBV infections (CHB), 47 resolved infections, and 56 non-infected controls, respectively. The rate of CD4+/CD8+ T cell proliferation in OBI or CHB carriers was higher than in HBV resolved and non-infected individuals (P < 0.05). The intensity of IFN-γ-secretion T-cell response of OBI carriers was highest, followed by CHB and resolved infections, and non-infected individuals (P < 0.05). The frequency of intracellular IFN-γ and IL-17A CD4+/CD8+ and IL-21 CD4+ T-cell responses was significantly higher in resolved infections than in OBI or CHB carriers (P < 0.05), while the level of extracellular IL-17A of peripheral blood mononuclear cells (PBMCs) was higher in OBI and CHB carriers than in resolved infections (P < 0.01). The frequency of intracellular IL-10 CD4+ T-cell response in CHB, OBI, and resolved infections was higher than in HBV non-infected individuals (P < 0.01). Intracellular IL-10 CD8+ T cell and extracellular IL-10 T-cell responses were higher in CHB than in OBI (P = 0.012) or HBV resolved infections (P < 0.01). In conclusion, the higher level of effective T-cell response with IFN-γ, IL-17A, and IL-21 contributes to resolved infection outcome, while higher levels of suppressive T-cell response with IL-10 result in HBV chronicity. OBI is an intermediary status between HBV resolved and chronic infections, in which IL-21 effector and IL-10 suppressor T-cell responses play an important role in directing the outcome of HBV infection.

A Self-Biomineralized Novel Adenovirus Vectored COVID-19 Vaccine for Boosting Immunization of Mice.

SARS-CoV-2 has caused more than 3.8 million deaths worldwide, and several types of COVID-19 vaccines are urgently approved for use, including adenovirus vectored vaccines. However, the thermal instability and pre-existing immunity have limited its wide applications. To circumvent these obstacles, we constructed a self-biomineralized adenovirus vectored COVID-19 vaccine (Sad23L-nCoV-S-CaP) by generating a calcium phosphate mineral exterior (CaP) based on Sad23L vector carrying the full-length gene of SARS-CoV-2 spike protein (S) under physiological condition. This Sad23L-nCoV-S-CaP vaccine was examined for its characteristics of structure, thermostability, immunogenicity and avoiding the problem of preexisting immunity. In thermostability test, Sad23L-nCoV-S-CaP could be stored at 4 °C for over 45 days, 26 °C for more than 8 days and 37 °C for approximately 2 days. Furthermore, Sad23L-nCoV-S-CaP induced higher level of S-specific antibody and T cell responses, and was not affected by the pre-existing anti-Sad23L immunity, suggesting it could be used as boosting immunization on Sad23L-nCoV-S priming vaccination. The boosting with Sad23L-nCoV-S-CaP vaccine induced high titers of 105.01 anti-S1, 104.77 anti-S2 binding antibody, 103.04 pseudovirus neutralizing antibody (IC50), and robust T-cell response of IFN-γ (1466.16 SFCs/106 cells) to S peptides, respectively. In summary, the self-biomineralization of the COVID-19 vaccine Sad23L-nCoV-S-CaP improved vaccine efficacy, which could be used in prime-boost regimen for prevention of SARS-CoV-2 infection in humans.

Prime-boost vaccination of mice and rhesus macaques with two novel adenovirus vectored COVID-19 vaccine candidates.

ABSTRACTCOVID-19 vaccines are being developed urgently worldwide. Here, we constructed two adenovirus vectored COVID-19 vaccine candidates of Sad23L-nCoV-S and Ad49L-nCoV-S carrying the full-length gene of SARS-CoV-2 spike protein. The immunogenicity of two vaccines was individually evaluated in mice. Specific immune responses were observed by priming in a dose-dependent manner, and stronger responses were obtained by boosting. Furthermore, five rhesus macaques were primed with 5 × 109 PFU Sad23L-nCoV-S, followed by boosting with 5 × 109 PFU Ad49L-nCoV-S at 4-week interval. Both mice and macaques well tolerated the vaccine inoculations without detectable clinical or pathologic changes. In macaques, prime-boost regimen induced high titers of 103.16 anti-S, 102.75 anti-RBD binding antibody and 102.38 pseudovirus neutralizing antibody (pNAb) at 2 months, while pNAb decreased gradually to 101.45 at 7 months post-priming. Robust T-cell response of IFN-γ (712.6 SFCs/106 cells), IL-2 (334 SFCs/106 cells) and intracellular IFN-γ in CD4+/CD8+ T cell (0.39%/0.55%) to S peptides were detected in vaccinated macaques. It was concluded that prime-boost immunization with Sad23L-nCoV-S and Ad49L-nCoV-S can safely elicit strong immunity in animals in preparation of clinical phase 1/2 trials.

Marmoset Viral Hepatic Inflammation Induced by Hepatitis C Virus Core Protein via IL-32.

Common marmosets infected with GB virus-B (GBV-B) chimeras containing hepatitis C virus (HCV) core and envelope proteins (CE1E2p7) developed more severe hepatitis than those infected with HCV envelope proteins (E1E2p7), suggesting that HCV core protein might be involved in the pathogenesis of viral hepatitis. The potential role of HCV core in hepatic inflammation was investigated. Six individual cDNA libraries of liver tissues from HCV CE1E2p7 or E1E2p7 chimera-infected marmosets (three animals per group) were constructed and sequenced. By differential expression gene analysis, 30 of 632 mRNA transcripts were correlated with the immune system process, which might be associated with hepatitis. A protein-protein interaction network was constituted by STRING database based on these 30 differentially expressed genes (DEGs), showing that IL-32 might play a central regulatory role in HCV core-related hepatitis. To investigate the effect of HCV core protein on IL-32 production, HCV core expressing and mock constructs were transfected into Huh7 cells. IL-32 mRNA and secretion protein were detected at significantly higher levels in cells expressing HCV core protein than in those without HCV core expression (P < 0.01 and P < 0.001, respectively). By KEGG enrichment analysis and using the specific signaling pathway inhibitor LY294002 for inhibition of PI3K, IL-32 expression was significantly reduced (P < 0.001). In conclusion, HCV core protein induces an increase of IL-32 expression via the PI3K pathway in hepatic cells, which played a major role in development of HCV-related severe hepatitis.

A high infectious simian adenovirus type 23 vector based vaccine efficiently protects common marmosets against Zika virus infection.

Zika virus (ZIKV) has spread in many countries or territories causing severe neurologic complications with potential fatal outcomes. The small primate common marmosets are susceptible to ZIKV, mimicking key features of human infection. Here, a novel simian adenovirus type 23 vector-based vaccine expressing ZIKV pre-membrane-envelope proteins (Sad23L-prM-E) was produced in high infectious titer. Due to determination of immunogenicity in mice, a single-dose of 3×108 PFU Sad23L-prM-E vaccine was intramuscularly inoculated to marmosets. This vaccine raised antibody titers of 104.07 E-specific and 103.13 neutralizing antibody (NAb), as well as robust specific IFN-γ secreting T-cell response (1,219 SFCs/106 cells) to E peptides. The vaccinated marmosets, upon challenge with a high dose of ZIKV (105 PFU) six weeks post prime immunization, reduced viremia by more than 100 folds, and the low level of detectable viral RNA (<103 copies/ml) in blood, saliva, urine and feces was promptly eliminated when the secondary NAb (titer >103.66) and T-cell response (>726 SFCs/106 PBMCs) were acquired 1-2 weeks post exposure to ZIKV, while non-vaccinated control marmosets developed long-term high titer of ZIKV (105.73 copies/ml) (P<0.05). No significant pathological lesions were observed in marmoset tissues. Sad23L-prM-E vaccine was detectable in spleen, liver and PBMCs at least 4 months post challenge. In conclusion, a prime immunization with Sad23L-prM-E vaccine was able to protect marmosets against ZIKV infection when exposed to a high dose of ZIKV. This Sad23L-prM-E vaccine is a promising vaccine candidate for prevention of ZIKV infection in humans.

A rapid strategy for constructing novel simian adenovirus vectors with high viral titer and expressing highly antigenic proteins applicable for vaccine development.

Adenoviral vectors have been widely used for the development of infectious disease vaccines. However, the challenge of human adenoviral vector rooted from the predominant adenovirus serotype 5 strain limiting its usefulness by the widespread pre-existing neutralizing antibodies in recipients. To circumvent this obstacle, we generated an ad-hoc adenovirus vector in human or primates. Here, a chimeric simian adenoviral vector Sad23 was constructed consisting in deleting of E1 and E3 regions of the full-length simian adenovirus serotype 23 genome (SAdV23) by Gibson assembly. To improve Sad23 virus propagating efficiency, the E4 region open reading frame 6 (orf6) was replaced by the corresponding element of human adenovirus type 5 (Ad5), designated Sad23L. The procedure for cloning this novel vector took a single week, and recombinant adenovirus was packaged with high titer in HEK293 cells. To verify the ability of this novel adenoviral vector to deliver foreign genes, Zika virus (ZIKV) prM-E genes were used as target genes for antigen expression. Recombinant adenoviruses Sad23L-prM-E, Sad23-prM-E and Ad5-prM-E were intramuscularly inoculated into Ad5-eGFP none pre-exposed or pre-exposed mice, and the immune response to ZIKV prM-E was compared between vectors. Sad23L-prM-E induced a fairly robust immune response and maintained immunogenicity in Ad5 pre-exposed mice, which suggested that Ad5 pre-existing immunity did not affect Sad23L-prM-E immunization. These preliminary results suggest that the proposed rapid strategy was effective in constructing a new adenoviral vector platform (Sad23 L) usable for the development of human vaccines.