anti-Ad Antibodies Research Articles

A causal multiomics study discriminates the early immune features of Ad5-vectored Ebola vaccine recipients

The vaccine-induced innate immune response is essential for the generation of an antibody response. To date, how Ad5-vectored vaccines are influenced by preexisting anti-Ad5 antibodies during activation of the early immune response remains unclear. Here, we investigated the specific alterations in GP1,2-specific IgG-related elements of the early immune response at the genetic, molecular, and cellular levels on days 0, 1, 3, and 7 after Ad5-EBOV vaccination. In a causal multiomics analysis, distinct early immune responses associated with GP1,2-specific IgG were observed in Ad5-EBOV recipients with a low level of preexisting anti-Ad5 antibodies. This study revealed the correlates of the Ad5-EBOV-induced IgG response and provided mechanistic evidence for overcoming preexisting Ad5 immunity during the administration of Ad5-vectored vaccines.

Effects of pre-existing anti-adenovirus antibodies on transgene expression levels and therapeutic efficacies of arming oncolytic adenovirus

Oncolytic adenoviruses (OAds), most of which are based on species C human adenovirus serotype 5 (Ad5) (OAd5), have recently received much attention as potential anticancer agents. High seroprevalence of anti-Ad5 neutralizing antibodies is a major hurdle for Ad5-based gene therapy. However, the impacts of anti-Ad5 neutralizing antibodies on OAd5-mediated transgene expression in the tumor and antitumor effects remain to be fully elucidated. In this study, we examined the impact of anti-Ad5 neutralizing antibodies on the OAd5-mediated antitumor effects and OAd5-mediated transgene expression. The luciferase expression of OAd-tAIB-Luc, which contains the cytomegalovirus promoter-driven luciferase gene, was inhibited in human cultured cells in the presence of human serum. Although the inhibitory effects of human serum possessing the low anti-Ad5 neutralizing antibody titers were overcome by long-term infection, the in vitro tumor cell lysis activities of OAd-tAIB-Luc were entirely attenuated by human serum containing the high titers of anti-Ad5 neutralizing antibodies. OAd-tAIB-Luc-mediated luciferase expression in the subcutaneous tumors 3 days after administration and tumor growth suppression levels following intratumoral administration were significantly lower in mice possessing the high titers of anti-Ad5 neutralizing antibodies, compared to those in control mice. These results suggested that pre-existing anti-Ad5 antibodies attenuated both transgene expression and potential antitumor effects of OAd5 following intratumoral administration.

Phase I, multicenter, open-label study of intravenous VCN-01 oncolytic adenovirus with or without nab-paclitaxel plus gemcitabine in patients with advanced solid tumors

BackgroundVCN-01 is an oncolytic adenovirus (Ad5 based) designed to replicate in cancer cells with dysfunctional RB1 pathway, express hyaluronidase to enhance virus intratumoral spread and facilitate chemotherapy and immune cells...

Induction of Th1 and Th2 in the protection against SARS-CoV-2 through mucosal delivery of an adenovirus vaccine expressing an engineered spike protein

A series of recombinant human type 5 adenoviruses that express the full-length or membrane-truncated spike protein (S) of SARS-CoV-2 (AdCoV2-S or AdCoV2-SdTM, respectively) was tested the efficacy against SARS-CoV-2 via intranasal (i.n.) or subcutaneous (s.c.) immunization in a rodent model. Mucosal delivery of adenovirus (Ad) vaccines could induce anti-SARS-CoV-2 IgG and IgA in the serum and in the mucosal, respectively as indicated by vaginal wash (vw) and bronchoalveolar lavage fluid (BALF). Serum anti-SARS-CoV-2 IgG but not IgA in the vw and BALF was induced by AdCoV2-S s.c.. Administration of AdCoV2-S i.n. was able to induce higher anti-SARS-CoV-2 binding and neutralizing antibody levels than s.c. injection. AdCoV2-SdTM i.n. induced a lower antibody responses than AdCoV2-S i.n.. Induced anti-S antibody responses by AdCoV2-S via i.n. or s.c. were not influenced by the pre-existing serum anti-Ad antibody. Novelty, S-specific IgG1 which represented Th2-mediated humoral response was dominantly induced in Ad i.n.-immunized serum in contrast to more IgG2a which represented Th1-mediated cellular response found in Ad s.c.-immunized serum. The activation of S-specific IFN-ɣ and IL-4 in splenic Th1 and Th2 cells, respectively, was observed in the AdCoV2-S i.n. and s.c. groups, indicating the Th1 and Th2 immunity were activated. AdCoV2-S and AdCoV2-SdTM significantly prevented body weight loss and reduced pulmonary viral loads in hamsters. A reduction in inflammation in the lungs was observed in AdCoV-S via i.n. or s.c.-immunized hamsters following a SARS-CoV-2 challenge. It correlated to Th1 cytokine but no inflammatory cytokines secretions found in AdCoV-S i.n. -immunized BALF. These results indicate that intranasal delivery of AdCoV2-S vaccines is safe and potent at preventing SARS-CoV-2 infections.

The majority of SARS-CoV-2-specific antibodies in COVID-19 patients with obesity are autoimmune and not neutralizing

Background/objectivesObesity decreases the secretion of SARS-CoV-2-specific IgG antibodies in the blood of COVID-19 patients. How obesity impacts the quality of the antibodies secreted, however, is not understood. Therefore, the objective of this study is to evaluate the presence of neutralizing versus autoimmune antibodies in COVID-19 patients with obesity.Subjects/methodsThirty serum samples from individuals who tested positive for SARS-CoV-2 infection by RT-PCR were collected from inpatient and outpatient settings. Of these, 15 were lean (BMI < 25) and 15 were obese (BMI ≥ 30). Control serum samples were from 30 uninfected individuals, age-, gender-, and BMI-matched, recruited before the current pandemic. Neutralizing and autoimmune antibodies were measured by ELISA. IgG autoimmune antibodies were specific for malondialdehyde (MDA), a marker of oxidative stress and lipid peroxidation, and for adipocyte-derived protein antigens (AD), markers of virus-induced cell death in the obese adipose tissue.ResultsSARS-CoV-2 infection induces neutralizing antibodies in all lean but only in few obese COVID-19 patients. SARS-CoV-2 infection also induces anti-MDA and anti-AD autoimmune antibodies more in lean than in obese patients as compared to uninfected controls. Serum levels of these autoimmune antibodies, however, are always higher in obese versus lean COVID-19 patients. Moreover, because the autoimmune antibodies found in serum samples of COVID-19 patients have been correlated with serum levels of C-reactive protein (CRP), a general marker of inflammation, we also evaluated the association of anti-MDA and anti-AD antibodies with serum CRP and found a positive association between CRP and autoimmune antibodies.ConclusionsOur results highlight the importance of evaluating the quality of the antibody response in COVID-19 patients with obesity, particularly the presence of autoimmune antibodies, and identify biomarkers of self-tolerance breakdown. This is crucial to protect this vulnerable population at higher risk of responding poorly to infection with SARS-CoV-2 than lean controls.

Neutralizing Antibodies against SARS-CoV-2, Anti-Ad5 Antibodies, and Reactogenicity in Response to Ad5-nCoV (CanSino Biologics) Vaccine in Individuals with and without Prior SARS-CoV-2.

This is the first study outside of clinical trials (phase I–III) evaluating the ability of the Ad5-nCoV vaccine to generate neutralizing antibodies and the factors associated with optimal or suboptimal response. In a longitudinal assay, 346 people (117 with prior COVID-19 and 229 without prior COVID-19) vaccinated with Ad5-nCoV were recruited. The percentage of neutralizing antibodies against SARS-CoV-2 (Surrogate Virus Neutralization Test) and antibodies against Ad5 (ADV-Ad5 IgG ELISA) were quantified pre and post-vaccination effects. The Ad5-nCoV vaccine induces higher neutralizing antibodies percentage in individuals with prior COVID-19 than those without prior COVID-19 (median [IQR]: 98% [97–98.1] vs. 72% [54–90], respectively; p < 0.0001). Furthermore, a natural infection (before vaccination) induces more neutralizing antibodies percentage than immunized individuals without prior COVID-19 (p < 0.01). No patient had vaccine-severe adverse effects. The age, antidepressant, and immunosuppressive treatments, reactogenicity, and history of COVID-19 are associated with impaired antibody production. The anti-Ad5 antibodies increased after 21 days of post-vaccination in all groups (p < 0.01). We recommend the application of a booster dose of Ad5-nCoV, especially for those individuals without previous COVID-19 infection. Finally, the induction of anti-Ad5 antibodies after vaccination should be considered if a booster with the same vaccine is planned.

Polyethylene glycol–modified DOTAP:cholesterol/adenovirus hybrid vectors have improved transduction efficiency and reduced immunogenicity

Despite the success of recombinant adenovirus serotype 5 (Ad5) and dioleoyltrimethylaminopropane:cholesterol (DOTAP:Chol) liposomes as viral and nonviral vectors in clinical trials, these vectors still suffer from drawbacks such as high immunogenicity and low transfection efficiency, respectively. To address these issues, some researchers have synthesized DOTAP:Chol/Ad5 hybrid vectors hoping to combine the unique features of the individual vectors. While this method shows promise, the limitations associated with Ad5 were not completely ameliorated. The objective of the present study was to use PEGylated liposomes to improve upon these previous studies, with the aim of maintaining high transduction efficiency and reducing the immune response. PEGylated DOTAP:Chol liposomes were synthesized at different molar ratios of polyethylene glycol (PEG) (0 to 0.04), and its effects on transduction efficiency and immunogenicity of Ad5 were studied. Coating of Ad5 with PEGylated liposomes was confirmed using transmission electron microscopy and dynamic light scattering, and these techniques indicated that the size of the complexes was ~ 140 nm. Transduction efficiency was enhanced nearly 100-fold in CAR-negative cells treated with PEGylated liposome/Ad5 complexes compared to unmodified Ad5. In addition, PEGylated liposome/Ad5 complexes significantly reduced the humoral and innate immune responses relative to native Ad5 as assessed by antiAd5 antibodies and IL-6 production by macrophage cells. Moreover, PEGylation decreased the cytotoxicity and hemolytic activity of the liposomes. In conclusion, the transduction efficiency, biocompatibility, and immunogenicity of DOTAP:Chol/Ad5 complexes were significantly improved by including an optimum amount of a PEG-lipid.

Immunological Analysis of the Hepatitis B Virus "a" Determinant Displayed on Chimeric Virus-Like Particles of Macrobrachium rosenbergii Nodavirus Capsid Protein Produced in Sf9 Cells.

Chimeric virus-like particles (VLPs) have been widely exploited for various purposes including their use as vaccine candidates, particularly due to their ability to induce stronger immune responses than VLPs consisting of single viral proteins. In the present study, VLPs of the Macrobrachium rosenbergii nodavirus (MrNV) capsid protein (Nc) displaying the hepatitis B virus “a” determinant (aD) were produced in Spodoptera frugiperda (Sf9) insect cells. BALB/c mice immunised with the purified chimeric Nc-aD VLPs elicited a sustained titre of anti-aD antibody, which was significantly higher than that elicited by a commercially available hepatitis B vaccine and Escherichia coli-produced Nc-aD VLPs. Immunophenotyping showed that the Sf9-produced Nc-aD VLPs induced proliferation of cytotoxic T-lymphocytes and NK1.1 natural killer cells. Furthermore, enzyme-linked immunospot (ELISPOT)analysis showed the presence of antibody-secreting memory B cells in the mice splenocytes stimulated with the synthetic aD peptide. The significant humoral, natural killer cell and memory B cell immune responses induced by the Sf9-produced Nc-aD VLPs suggest that they present good prospects for use as a hepatitis B vaccine candidate.

Transient immunosuppression blocks iBALT formation in the lung induced by helper-dependent adenoviral vector during gene delivery

Abstract Lung is an immune sensitive organ with presence of all immune cells at the frontline of immunity. Under chronic infection conditions, inducible Bronchus-associated lymphoid tissue (iBALT) can be formed to defend foreign infectious organisms. We wonder whether viral vectors for gene therapy could induce iBALT formation and whether this process can be controlled through immune modulation. To test these ideas, we repeatedly administered a helper-dependent adenoviral (HD-Ad) vector to mouse lungs and examined iBALT formation. HD-Ad vectors are based on adenovirus but with all viral coding sequences deleted and is one option for large gene delivery. In our previous studies, HD-Ad vectors have exhibited high in vivo efficiency in transduction of mouse, rabbit and pig airway epithelial cells, including stem/progenitor cells such as basal cells. HD-Ad vectors expressing therapeutic gene encoding cystic fibrosis transmenbrane conductance regulator (CFTR) rescue CFTR function in CF patient primary cells. In our mouse studies, we found that HD-Ad vectors induced iBALT formation and antibody production. We used cyclophosphamide or HD-Ad expressing IL-10 to modulate the host immune responses and to examine the effects on iBALT formation. Comparing with control group (HD-Ad vectors only), the mice treated with cyclophosphamide or IL-10 expressing HD-Ad vector showed a significant reduction of iBALT formation and infiltration of inflammatory cells, including CD4+ and B220+ as well as production of anti-Ad antibodies and neutralizing antibodies. Our results indicate that transient modulation significantly mitigated immunopathology and extended transgene expression in mouse airways following readministration of HD-Ad vectors.

Semi-quantification of antibody-dependent enhancement (ADE) in the uptake of Adenovirus serotype 5 into THP-1 cells

Replication defective recombinant Ad5 vectors (rAdV5) are extensively explored for its applications in gene therapy and vaccine delivery. Ad5 enter into monocytes and macrophages through CAR independent route as an immune complex termed as antibody-dependent enhancement (ADE). We developed an effective method for estimating the ADE of rAdV5 encoding GFP (rAdV5-GFP) into THP-1 cells, using fluorimetric semi-quantification of GFP. Initially, twenty numbers of human sera samples were screened in HeLa cells for anti-Ad5 antibody titer using neutralization assay. Uptake of rAdV5-GFP in THP-1 cells was observed only after pre-incubation with the serially diluted human sera which are attributed to ADE. The optimal dilution which showed the maximum GFP expression as per the fluorescence microscopic analysis in THP-1 cells was used for further analysis. Fluorimetric analysis of the THP-1 cell lysate showed a maximum GFP intensity of 17058 RFU, which was equivalent to the 0.397 pmoles of Alexa Fluor 488 under the same experimental condition. Similarly, immunoblot analysis of GFP in THP-1 cell lysate and HeLa cell lysate confirmed the entry of rAdV5-GFP into the cells. The assay can serve as a platform for understanding the molecular events involved in ADE for the uptake of viruses into immune cells.

Oral immunization of a rAd vector expressing norovirus VP1 elicits a potent mucosal immune response without an increase in anti-vector immunity

Background: Noroviruses are the leading cause of epidemic acute gastroenteritis and foodborne diarrheal disease in humans. Potential correlates of protection identified through human challenge studies include mucosal IgA, memory B cells, and serum blocking (BT50) antibody titers. We hypothesized that fecal and memory IgA responses would be significantly elevated following oral immunization. Methods & Materials: We conducted a single-site, randomized, double-blind, placebo-controlled trial to determine the safety and immunogenicity of a norovirus vaccine consisting of a non-replicating adenovirus vector expressing VP1 delivered orally by tablet. Subjects that met inclusion/exclusion criteria were enrolled and randomized 2:1 to receive either a single dose of vaccine or placebo, respectively. Two dose levels were evaluated. The primary objective was to assess safety. The secondary objective was to determine immunogenicity, primarily assessed by measuring serum BT50. Exploratory immunological assessments included serum, fecal and saliva antibody titers measured by and the frequency of memory and antibody secreting B cells (ASCs). Anti-vector immunity was assessed by measuring Ad5 neutralizing antibody responses pre and post immunization (ClinicalTrials.gov number NCT02868073). Results: Sixty-six subjects were enrolled between July and Sept 2016. The vaccine was well-tolerated. The primary immunological endpoint (BT50 titers) was met in low and high dose groups (P = 0.0014, P < 0.0001). In the high-dose group, 18/23 (78%) subjects had a two-fold or greater increase in BT50 titer after a single vaccine dose. An average of 561 IgA ASCs x106 PBMC were induced post-immunization in the high dose group. Post immunization, a 15 fold increase in the geometric mean was observed, along with a greater than 10 fold average increase in the fecal IgA VP1 specific antibody response. Anti-Ad5 neutralizing antibody responses did not significantly increase compared to the placebo group. Conclusion: The norovirus vaccine was well-tolerated (with no dose limiting toxicities), and generated immune responses to norovirus VP1, without a corresponding increase in anti-Ad5 neutralizing antibody responses. It particular, the vaccine generated memory and local effector IgA + B cells. Because mucosal IgA is the front-line defense against enteric pathogens, vaccine-derived norovirus-specific intestinal IgA may translate to superior efficacy. This is a very promising advance in the development of an oral norovirus vaccine.

Immunogenicity of adenovirus-vector vaccine targeting hepatitis B virus: non-clinical safety assessment in non-human primates

BackgroundA new promising therapeutic approach has emerged for patients chronically infected by the hepatitis B virus (HBV) with the development of a non-replicative adenovirus vector vaccine candidate (Ad-HBV). The vaccine encodes a fusion protein composed of a truncated HBV core protein, mutated polymerase protein, and two envelope domains. In this study, we assessed the immunogenicity of Ad-HBV administered to cynomolgus monkeys during a non-clinical safety assessment.MethodsThe virus was subcutaneously administered at 1.0 × 109 viral particles (VP)/animal (low-dose group), 1.0 × 1010 VP/animal (mid-dose group), and 1.0 × 1011 VP/animal (high-dose group); the control groups were administered an Ad5-null virus (1.0 × 1011 VP/animal) and saline only.ResultsExcept for inflammatory cell infiltration under the skin at the injection sites and transient elevation of body temperature and serum albumin, no Ad-HBV-related toxic effects were noted in any treatment group. Moreover, interferon (IFN)-γ enzyme-linked immunospot assays showed that Ad-HBV induced the targeting of T cells to a broad spectrum of HBV-specific epitopes spanning all three of the selected HBV immunogens (core, polymerase, and envelope domains) in a dose-dependent manner. Although anti-Ad antibody was produced in all groups (except for the saline control), the antibody titers were significantly lower in the high-dose Ad-HBV group than in the group that received the same dose of the Ad-null empty vector. In addition, the IFN-γ and IL-2 expression levels in the liver were significantly improved for the mid-dose, high-dose, and Ad-null control group (p < 0.05), but not for the low-dose group.ConclusionsTaken together, this safety assessment indicates that the Ad-HBV candidate vaccine is a potent specific immunotherapeutic agent, supporting its further clinical development as an anti-HBV infection vaccine.

Scavenger receptor-mediated Ad5 entry and acLDL accumulation in monocytes/macrophages synergistically trigger innate responses against viral infection

Adenovirus serotype 5 (Ad5) is a common cause of respiratory tract infection, and populations worldwide have high prevalence of anti-Ad5 antibodies, implying extensively prior infection. Ad5 infection potently activates the host innate defense and inflammation, but the molecular mechanisms are not completely clarified. We report here that monocytes from Ad5-seropositive subjects upregulates the expression of scavenger receptor A (SR-A), and the increased SR-A promote the susceptibility of Ad5 entry and subsequent innate signaling activation. SR-A is also known as major receptor for lipid uptake, we therefore observed that monocytes from Ad5-seropositive subjects accumulated the acetylated low-density lipoprotein (acLDL) and had the elevated cellular stress to induce the activation of monocyte/macrophages. These findings demonstrate that SR-A-mediated Ad5 entry, innate signaling activation and acLDL accumulation synergistically trigger the robust antiviral innate and inflammatory responses, which are helpful to our understanding of the pathogenesis of adenovirus infection.

A prime-boost immunization regimen based on a simian adenovirus 36 vectored multi-stage malaria vaccine induces protective immunity in mice

Malaria remains a considerable burden on public health. In 2015, the WHO estimates there were 212 million malaria cases causing nearly 429,000 deaths globally. A highly effective malaria vaccine is needed to reduce the burden of this disease. We have developed an experimental vaccine candidate (PyCMP) based on pre-erythrocytic (CSP) and erythrocytic (MSP1) stage antigens derived from the rodent malaria parasite P. yoelii. Our protein-based vaccine construct induces protective antibodies and CD4+ T cell responses. Based on evidence that viral vectors increase CD8+ T cell-mediated immunity, we also have tested heterologous prime-boost immunization regimens that included human adenovirus serotype 5 vector (Ad5), obtaining protective CD8+ T cell responses. While Ad5 is commonly used for vaccine studies, the high prevalence of pre-existing immunity to Ad5 severely compromises its utility. Here, we report the use of the novel simian adenovirus 36 (SAd36) as a candidate for a vectored malaria vaccine since this virus is not known to infect humans, and it is not neutralized by anti-Ad5 antibodies. Our study shows that the recombinant SAd36PyCMP can enhance specific CD8+ T cell response and elicit similar antibody titers when compared to an immunization regimen including the recombinant Ad5PyCMP. The robust immune responses induced by SAd36PyCMP are translated into a lower parasite load following P. yoelii infectious challenge when compared to mice immunized with Ad5PyCMP.

Native and engineered tropism of vectors derived from a rare species D adenovirus serotype 43.

Unique molecular properties of species D adenoviruses (Ads)—the most diverse yet underexplored group of Ads—have been used to develop improved gene vectors. The low seroprevalence in humans of adenovirus serotype 43 (Ad43), an otherwise unstudied species D Ad, identified this rare serotype as an attractive new human gene therapy vector platform. Thus, in this study we wished to assess biological properties of Ad43 essential to its vectorization. We found that (1) Ad43 virions do not bind blood coagulation factor X and cause low random transduction upon vascular delivery; (2) they clear host tissues more quickly than do traditionally used Ad5 vectors; (3) Ad43 uses CD46 as primary receptor; (4) Ad43 can use integrins as alternative primary receptors. As the first step toward vectorization of Ad43, we demonstrated that the primary receptor specificity of the Ad43 fiber can be altered to achieve infection via Her2, an established oncotarget. Whereas this modification required use of the Ad5 fiber shaft, the presence of this domain in chimeric virions did not make them susceptible for neutralization by anti-Ad5 antibodies.

Diminished Innate Antiviral Response to Adenovirus Vectors in cGAS/STING-Deficient Mice Minimally Impacts Adaptive Immunity.

Infection by adenovirus, a nonenveloped DNA virus, induces antiviral innate and adaptive immune responses. Studies of transformed human and murine cell lines using short hairpin RNA (shRNA) knockdown strategies identified cyclic guanine adenine synthase (cGAS) as a pattern recognition receptor (PRR) that contributes to the antiadenovirus response. Here we demonstrate how the cGAS/STING cascade influences the antiviral innate and adaptive immune responses in a murine knockout model. Using knockout bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophages (BMMOs), we determined that cGAS and STING are essential to the induction of the antiadenovirus response in these antigen-presenting cells (APCs) in vitro We next determined how the cGAS/STING cascade impacts the antiviral response following systemic administration of a recombinant adenovirus type 5 vector (rAd5V). Infection of cGAS(-/-) and STING(-/-) mice results in a compromised early antiviral innate response compared to that in wild-type (WT) controls: significantly lower levels of beta interferon (IFN-β) secretion, low levels of proinflammatory chemokine induction, and reduced levels of antiviral transcript induction in hepatic tissue. At 24 h postinfection, levels of viral DNA and reporter gene expression in the liver were similar in all strains. At 28 days postinfection, clearance of infected hepatocytes in cGAS or STING knockout mice was comparable to that in WT C57BL/6 mice. Levels of neutralizing anti-Ad5V antibody were modestly reduced in infected cGAS mice. These data support a dominant role for the cGAS/STING cascade in the early innate antiviral inflammatory response to adenovirus vectors. However, 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. The detection of viral infection by host sentinel immune cells contributes to the activation of a complex and varied antiviral innate and adaptive immune response, which limits virus replication, spread, and susceptibility to infection. In this study, we have characterized how the cGAS/STING DNA-sensing cascade contributes to early detection of adenovirus infections. cGAS influences APC activation and early innate antiviral inflammatory immune responses, but adaptive immune pathways associated with virus clearance and anti-Ad antibody production were minimally influenced by the loss of the cGAS PRR signaling cascade.

111. Efficient Transduction in Neonatal Mice via Systemic Administration of an Adenovirus Vector

An adenovirus (Ad) vector is widely used as a gene delivery vector due to their various advantages. An Ad vector is applicable to innate genetic diseases, which are crucial targets of gene therapy. Patients of several innate genetic disorders often display symptoms in their early stage of life and are danger of death without any treatments. Radical treatments in infant ages are required in such patients. Gene therapy in neonates and infants is promising for such innate genetic disorders. In addition, although immune responses to an Ad vector hamper Ad vector-mediated transduction in adults, it is expected that such inhibitions do not occur in neonates and infants because of their immature immunity; however, transduction properties of Ad vectors in neonates and infants have not been fully examined. In this study, Ad vector-mediated transduction properties in neonatal mice were examined.A first-generation Ad vector containing a CMV promoter-driven firefly luciferase expression cassette was administered to newborn mice on the second day of life at a dose of 5.9×1011 infectious unit (IFU)/kg (this titer is equal to 1010 IFU/5-weeks old mice) via retro-orbital sinus. First, biodistribution of an Ad vector in neonatal mice was investigated 2 days after administration. As is the case with adult mice, the highest copy numbers of Ad vector genome were found in the liver among the organs. The liver also showed the highest levels of luciferase expression among the organs in neonates, although the transduction efficiency in the neonatal liver was approximately 5-fold lower than that in the adult liver. On the other hand, neonatal mice exhibited 10-fold higher levels of luciferase expression in the heart than adult mice. While approximately more than 1000-fold higher transduction efficiency was found in the adult liver, compared with the adult heart, the neonatal liver exhibited only 30-fold higher transduction efficiency than the neonatal heart. In order to achieve liver-specific transduction, a liver-specific promoter (a fusion promoter composed of apolipoprotein E enhancer, the hepatocyte control region, and human alpha 1-antitrypsin promoter) was incorporated into an Ad vector. The Ad vector containing the liver-specific promoter mediated almost 100-fold higher transduction efficiencies in the liver, compared with those in the other organs. Second, Ad vector-induced hepatotoxicity in neonatal mice was evaluated. Obvious elevation in serum alanine aminotransferase (ALT) activity in neonatal mice was not observed within 2 weeks following Ad vector administration, indicating that this dose of Ad vectors did not provoke significant hepatotoxicity in neonatal mice. In addition, while anti-Ad antibodies were strongly induced in the serum of adult mice on 21 days post Ad vector injection, almost background levels of anti-Ad antibodies were detected in neonatal mice. These results provide crucial information for gene therapy in neonates and infants.

Bolstering Components of the Immune Response Compromised by Prior Exposure to Adenovirus: Guided Formulation Development for a Nasal Ebola Vaccine.

The severity and longevity of the current Ebola outbreak highlight the need for a fast-acting yet long-lasting vaccine for at-risk populations (medical personnel and rural villagers) where repeated prime-boost regimens are not feasible. While recombinant adenovirus (rAd)-based vaccines have conferred full protection against multiple strains of Ebola after a single immunization, their efficacy is impaired by pre-existing immunity (PEI) to adenovirus. To address this important issue, a panel of formulations was evaluated by an in vitro assay for their ability to protect rAd from neutralization. An amphiphilic polymer (F16, FW ∼39,000) significantly improved transgene expression in the presence of anti-Ad neutralizing antibodies (NAB) at concentrations of 5 times the 50% neutralizing dose (ND50). In vivo performance of rAd in F16 was compared with unformulated virus, virus modified with poly(ethylene) glycol (PEG), and virus incorporated into poly(lactic-co-glycolic) acid (PLGA) polymeric beads. Histochemical analysis of lung tissue revealed that F16 promoted strong levels of transgene expression in naive mice and those that were exposed to adenovirus in the nasal cavity 28 days prior to immunization. Multiparameter flow cytometry revealed that F16 induced significantly more polyfunctional antigen-specific CD8+ T cells simultaneously producing IFN-γ, IL-2, and TNF-α than other test formulations. These effects were not compromised by PEI. Data from formulations that provided partial protection from challenge consistently identified specific immunological requirements necessary for protection. This approach may be useful for development of formulations for other vaccine platforms that also employ ubiquitous pathogens as carriers like the influenza virus.

Immune-correlates analysis of an HIV-1 vaccine efficacy trial reveals an association of nonspecific interferon-γ secretion with increased HIV-1 infection risk: a cohort-based modeling study.

BackgroundElevated risk of HIV-1 infection among recipients of an adenovirus serotype 5 (Ad5)-vectored HIV-1 vaccine was previously reported in the Step HIV-1 vaccine efficacy trial. We assessed pre-infection cellular immune responses measured at 4 weeks after the second vaccination to determine their roles in HIV-1 infection susceptibility among Step study male participants.MethodsWe examined ex vivo interferon-γ (IFN-γ) secretion from peripheral blood mononuclear cells (PBMC) using an ELISpot assay in 112 HIV-infected and 962 uninfected participants. In addition, we performed flow cytometric assays to examine T-cell activation, and ex vivo IFN-γ and interleukin-2 secretion from CD4+ and CD8+ T cells. We accounted for the sub-sampling design in Cox proportional hazards models to estimate hazard ratios (HRs) of HIV-1 infection per 1-loge increase of the immune responses.FindingsWe found that HIV-specific immune responses were not associated with risk of HIV-1 infection. However, each 1-loge increase of mock responses measured by the ELISpot assay (i.e., IFN-γ secretion in the absence of antigen-specific stimulation) was associated with a 62% increase of HIV-1 infection risk among vaccine recipients (HR = 1.62, 95% CI: (1.28, 2.04), p<0.001). This association remains after accounting for CD4+ or CD8+ T-cell activation. We observed a moderate correlation between ELISpot mock responses and CD4+ T-cells secreting IFN-γ (ρ = 0.33, p = 0.007). In addition, the effect of the Step vaccine on infection risk appeared to vary with ELISpot mock response levels, especially among participants who had pre-existing anti-Ad5 antibodies (interaction p = 0.04).ConclusionsThe proportion of cells, likely CD4+ T-cells, producing IFN-γ without stimulation by exogenous antigen appears to carry information beyond T-cell activation and baseline characteristics that predict risk of HIV-1 infection. These results motivate additional investigation to understand the potential link between IFN-γ secretion and underlying causes of elevated HIV-1 infection risk among vaccine recipients in the Step study.

Comparative analysis of immunization schedules using a novel adenovirus-based immunotherapeutic targeting hepatitis B in naïve and tolerant mouse models

Development of active targeted immunotherapeutics is a rapid developing field in the arena of chronic infectious diseases. The question of repeated, closely spaced administration of immunotherapeutics to achieve a rapid impact on the replicating agent is an important one. We analyzed here, using a prototype adenovirus-based immunotherapeutic encoding Core and Polymerase from the hepatitis B virus (Ad-HBV), the influence of closely spaced repeated immunizations on the level and quality of induced HBV-specific and vector-specific immune responses in various mouse models. Ad-HBV, whether injected once or multiple times, was able to induce HBV- and adeno-specific T cells both in HBV-free mice and in a HBV tolerant mouse model. Adenovirus-specific T cell responses and titers of neutralizing anti-Ad5 antibodies increased from time of the 3rd injection. Interestingly, single or multiple Ad-HBV injections resulted in detection of Polymerase-specific functional T cells in HBV tolerant mice. Overall no modulation of the levels of HBV-specific cytokine-producing (IFNγ/TNFα) and cytolytic T cells was observed following repeated administrations (3 or 6 weekly injections) when compared with levels detected after a single injection with the exception of two markers: 1. the proportion of HBV-specific IFNγ-producing cells bearing the CD27+/CD43+ phenotype appeared to be sustained in C57BL/6J mice following 6 weekly injections; 2. the percentage of IFNγ/TNFα Core-specific producing cells observed in spleens of HLA-A2 mice as well as of that specific of Polymerase observed in livers of HBV tolerant mice was maintained. In addition, percentage of HBV-specific T cells expressing PD-1 was not increased by multiple injections. Overall these data show that, under experimental conditions used, rapid, closely spaced administrations of an adenovirus-based HBV immunotherapeutics does not inhibit induced T-cell responses including in a HBV-tolerant environment.