Simian Adenovirus Research Articles

Expanding the Scope of Adenoviral Vectors by Utilizing Novel Tools for Recombination and Vector Rescue.

Recombinant adenoviruses are widely used in clinical and laboratory applications. Despite the wide variety of available sero- and genotypes, only a fraction is utilized in vivo. As adenoviruses are a large group of viruses, displaying many different tropisms, immune epitopes, and replication characteristics, the merits of translating these natural benefits into vector applications are apparent. This translation, however, proves difficult, since while research has investigated the application of these viruses, there are no universally applicable rules in vector design for non-classical adenovirus types. In this paper, we describe a generalized workflow that allows vectorization, rescue, and cloning of all adenoviral species to enable the rapid development of new vector variants. We show this using human and simian adenoviruses, further modifying a selection of them to investigate their gene transfer potential and build potential vector candidates for future applications.

Redirect Tropism of Fowl Adenovirus 4 Vector by Modifying Fiber2 with Variable Domain of Heavy-Chain Antibody.

The variable domain of a heavy-chain antibody (VHH) has the potential to be used to redirect the cell tropism of adenoviral vectors. Here, we attempted to establish platforms to simplify the screening of VHHs for their specific targeting function when being incorporated into the fiber of adenovirus. Both fowl adenovirus 4 (FAdV-4) and simian adenovirus 1 (SAdV-1) have two types of fiber, one of which is dispensable for virus propagation and is a proper site for VHH display. An intermediate plasmid, pMD-FAV4Fs, was constructed as the start plasmid for FAdV-4 fiber2 modification. Foldon from phage T4 fibritin, a trigger for trimerization, was employed to bridge the tail/shaft domain of fiber2 and VHHs against human CD16A, a key membrane marker of natural killer (NK) cells. Through one step of restriction-assembly, the modified fiber2 was transferred to the adenoviral plasmid, which was linearized and transfected to packaging cells. Five FAdV-4 viruses carrying the GFP gene were finally rescued and amplified, with three VHHs being displayed. One recombinant virus, FAdV4FC21-EG, could hardly transduce human 293 or Jurkat cells. In contrast, when it was used at a multiplicity of infection of 1000 viral particles per cell, the transduction efficiency reached 51% or 34% for 293 or Jurkat cells expressing exogenous CD16A. Such a strategy of fiber modification was transplanted to the SAdV-1 vector to construct SAdV1FC28H-EG, which moderately transduced primary human NK cells while the parental virus transduced none. Collectively, we reformed the strategy of integrating VHH to fiber and established novel platforms for screening VHHs to construct adenoviral vectors with a specific tropism.

The Biodistribution of Replication-Defective Simian Adenovirus 1 Vector in a Mouse Model.

The administration route affects the biodistribution of a gene transfer vector and the expression of a transgene. A simian adenovirus 1 vector carrying firefly luciferase and GFP reporter genes (SAdV1-GFluc) were constructed, and its biodistribution was investigated in a mouse model by bioluminescence imaging and virus DNA tracking with real-time PCR. Luciferase activity and virus DNA were mainly found in the liver and spleen after the intravenous administration of SAdV1-GFluc. The results of flow cytometry illustrated that macrophages in the liver and spleen as well as hepatocytes were the target cells. Repeated inoculation was noneffective because of the stimulated serum neutralizing antibodies (NAbs) against SAdV-1. A transient, local expression of low-level luciferase was detected after intragastric administration, and the administration could be repeated without compromising the expression of the reporter gene. Intranasal administration led to a moderate, constant expression of a transgene in the whole respiratory tract and could be repeated one more time without a significant increase in the NAb titer. An immunohistochemistry assay showed that respiratory epithelial cells and macrophages in the lungs were transduced. High luciferase activity was restricted at the injection site and sustained for a week after intramuscular administration. A compromised transgene expression was observed after a repeated injection. When these mice were intramuscularly injected for a third time with the human adenovirus 5 (HAdV-5) vector carrying a luciferase gene, the luciferase activity recovered and reached the initial level, suggesting that the sequential use of SAdV-1 and HAdV-5 vectors was practicable. In short, the intranasal inoculation or intramuscular injection may be the preferred administration routes for the novel SAdV-1 vector in vaccine development.

Isolation of a recombinant simian adenovirus encoding the human adenovirus G52 hexon suggests a simian origin for human adenovirus G52.

Human adenoviruses (HAdVs) are causative agents of morbidity and mortality throughout the world. These double-stranded DNA viruses are phylogenetically classified into seven different species (A-G). HAdV-G52, originally isolated in 2008 from a patient presenting with gastroenteritis, is the sole human-derived member of species G. Phylogenetic analysis previously suggested that HAdV-G52 may have a simian origin, indicating a potential zoonotic spillover into humans. However, evidence of HAdV-G52 in either human or simian populations has not been reported since. Here, we describe the isolation and in vitro characterization of rhesus (rh)AdV-69, a novel simian AdV with clear evidence of recombination with HAdV-G52, from the stool of a rhesus macaque. Specifically, the rhAdV-69 hexon capsid protein is 100% identical to that of HAdV-G52, whereas the remainder of the genome is most similar to rhAdV-55, sharing 95.36% nucleic acid identity. A second recombination event with an unknown adenovirus (AdV) is evident at the short fiber gene. From the same sample, we also isolated a second, highly related recombinant AdV (rhAdV-68) that harbors a distinct hexon gene but nearly identical backbone compared to rhAdV-69. In vitro, rhAdV-68 and rhAdV-69 demonstrate comparable growth kinetics and tropisms in human cell lines, nonhuman cell lines, and human enteroids. Furthermore, we show that coinfection of highly related AdVs is not unique to this sample since we also isolated coinfecting rhAdVs from two additional rhesus macaque stool samples. Our data collectively contribute to elucidating the origins of HAdV-G52 and provide insights into the frequency of coinfections and subsequent recombination in AdV evolution.IMPORTANCEUnderstanding the host origins of adenoviruses (AdVs) is critical for public health as transmission of viruses from animals to humans can lead to emergent viruses. Recombination between animal and human AdVs can also produce emergent viruses. HAdV-G52 is the only human-derived member of the HAdV G species. It has been suggested that HAdV-G52 has a simian origin. Here, we isolated from a rhesus macaque, a novel rhAdV, rhAdV-69, that encodes a hexon protein that is 100% identical to that of HAdV-G52. This observation suggests that HAdV-G52 may indeed have a simian origin. We also isolated a highly related rhAdV, differing only in the hexon gene, from the same rhesus macaque stool sample as rhAdV-69, illustrating the potential for co-infection of closely related AdVs and recombination at the hexon gene. Furthermore, our study highlights the critical role of whole-genome sequencing in understanding AdV evolution and monitoring the emergence of pathogenic AdVs.

A Bivalent Adenovirus-Vectored Vaccine Induces a Robust Humoral Response, but Does Not Protect Cynomolgus Macaques Against a Lethal Challenge With Sudan Virus.

The most recent Sudan virus (SUDV) outbreak in Uganda was first detected in September 2022 and resulted in 164 laboratory-confirmed cases and 77 deaths. There are no approved vaccines against SUDV. Here, we investigated the protective efficacy of ChAdOx1-biEBOV in cynomolgus macaques using a prime or a prime-boost regimen. ChAdOx1-biEBOV is a replication-deficient simian adenovirus vector encoding SUDV and Ebola virus (EBOV) glycoproteins (GPs). Intramuscular vaccination induced SUDV and EBOV GP-specific immunoglobulin G responses and neutralizing antibodies. Upon challenge with SUDV, vaccinated animals showed signs of disease like those observed in control animals, and no differences in survival outcomes were measured among all 3 groups. Viral load in blood samples and in tissue samples obtained after necropsy were not significantly different between groups. Overall, this study highlights the importance of evaluating vaccines in multiple animal models and demonstrates the importance of understanding protective efficacy in both animal models and human hosts.

Markers of antroponotic viral infections in vervet monkeys arrived from their natural habitat (Tanzania).

Various human viruses have been identified in wild monkeys and in captive primates. Cases of transmission of viruses from wild monkeys to humans and vice versa are known. The aim of this study was to identify markers of anthroponotic viral infections in vervet monkeys (Chlorocebus pygerythrus) arrived from their natural habitat (Tanzania). Fecal samples (n = 56) and blood serum samples (n = 75) obtained from 75 animals, respectively, on days 10 and 23 after admission to the primate center, were tested for the markers of anthroponotic viral infections (Ebola virus, Marburg virus, lymphocytic choriomeningitis, hepatitis C virus, herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), parainfluenza types 1 and 3, intestinal adenoviruses, rotaviruses) by enzyme immunoassay (ELISA) and polymerase chain reaction (PCR). Among the examined animals, markers of 6 out of 11 tested viral infections were identified. Detection rates of IgG antibodies to HSV-1,2 (15.9%) and CMV (15.9%) were two times as low as IgG antibodies to EBV (31.8%). Among the markers of respiratory viral infections, IgG antibodies to parainfluenza virus type 1 were found (6.8%). 14.3% of the animals had rotavirus antigen, and 94% had simian adenovirus DNA. Markers of hemorrhagic fevers Ebola, Marburg, LCM, hepatitis C, and type 3 parainfluenza were not detected. When importing monkeys from different regions of the world, an expanded screening for viral infections is needed considering the epidemiological situation both in the country of importation and in the country of destination.

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.

Isolation of novel simian adenoviruses from macaques for development of a vector for human gene therapy and vaccines.

Adenoviruses are widely used in gene therapy and vaccine delivery. Due to the high prevalence of human adenoviruses (HAdVs), the pre-existing immunity against HAdVs in humans is common, which limits the wide and repetitive use of HAdV vectors. In contrast, the pre-existing immunity against simian adenoviruses (SAdVs) is low in humans. Therefore, we performed epidemiological investigations of SAdVs in simians and found that the SAdV prevalence was as high as 33.9%. The whole-genome sequencing and sequence analysis showed SAdV diversity and possible cross species transmission. One isolate with low level of pre-existing neutralizing antibodies in humans was used to construct replication-deficient SAdV vectors with E4orf6 substitution and E1/E3 deletion. Interestingly, we found that the E3 region plays a critical role in its replication in human cells, but the absence of this region could be compensated for by the E4orf6 from HAdV-5 and the E1 expression intrinsic to HEK293 cells.

Combined intranasal and intramuscular parainfluenza 5-, simian adenovirus ChAdOx1- and poxvirus MVA-vectored vaccines induce synergistically HIV-1-specific T cells in the mucosa.

The primary goal of this work is to broaden and enhance the options for induction of protective CD8+ T cells against HIV-1 and respiratory pathogens. We explored the advantages of the parainfluenza virus 5 (PIV5) vector for delivery of pathogen-derived transgenes alone and in combination with the in-human potent regimen of simian adenovirus ChAdOx1 prime-poxvirus MVA boost delivering bi-valent mosaic of HIV-1 conserved regions designated HIVconsvX. We showed in BALB/c mice that the PIV5 vector expressing the HIVconsvX immunogens could be readily incorporated with the other two vaccine modalities into a single regimen and that for specific vector combinations, mucosal CD8+ T-cell induction was enhanced synergistically by a combination of the intranasal and intramuscular routes of administration. Encouraging safety and immunogenicity data from phase 1 human trials of ChAdOx1- and MVA-vectored vaccines for HIV-1, and PIV5-vectored vaccines for SARS-CoV-2 and respiratory syncytial virus pave the way for combining these vectors for HIV-1 and other indications in 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/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.

A Novel Simian Adenovirus Associating with Human Adeno-virus Species G Isolated from Long-Tailed Macaque Feces.

Metagenomics has demonstrated its capability in outbreak investigations and pathogen surveillance and discovery. With high-throughput and effective bioinformatics, many disease-causing agents, as well as novel viruses of humans and animals, have been identified using metagenomic analysis. In this study, a VIDISCA metagenomics workflow was used to identify potential unknown viruses in 33 fecal samples from asymptomatic long-tailed macaques (Macaca fascicularis) in Ratchaburi Province, Thailand. Putatively novel astroviruses, enteroviruses, and adenoviruses were detected and confirmed by PCR analysis of long-tailed macaque fecal samples collected from areas in four provinces, Ratchaburi, Kanchanaburi, Lopburi, and Prachuap Khiri Khan, where humans and monkeys live in proximity (total n = 187). Astroviruses, enteroviruses, and adenoviruses were present in 3.2%, 7.5%, and 4.8% of macaque fecal samples, respectively. One adenovirus, named AdV-RBR-6-3, was successfully isolated in human cell culture. Whole-genome analysis suggested that it is a new member of the species Human adenovirus G, closely related to Rhesus adenovirus 53, with evidence of genetic recombination and variation in the hexon, fiber, and CR1 genes. Sero-surveillance showed neutralizing antibodies against AdV-RBR-6-3 in 2.9% and 11.2% of monkeys and humans, respectively, suggesting cross-species infection of monkeys and humans. Overall, we reported the use of metagenomics to screen for possible new viruses, as well as the isolation and molecular and serological characterization of the new adenovirus with cross-species transmission potential. The findings emphasize that zoonotic surveillance is important and should be continued, especially in areas where humans and animals interact, to predict and prevent the threat of emerging zoonotic pathogens.

Characterization of monkey adenoviruses with three fiber genes.

Although the occurrence of three fiber genes in monkey adenoviruses had already been described, the relatedness of the "extra" fibers have not yet been discussed. Here we report the genome analysis of two simian adenovirus (SAdV) serotypes from Old World monkeys and the phylogenetic analysis of the multiple fiber genes found in these and related AdVs. One of the newly sequenced serotypes (SAdV-2), isolated from a rhesus macaque (Macaca mulatta), was classified into species Human mastadenovirus G (HAdV-G), while the other serotype (SAdV-17), originating from a grivet (Chlorocebus aethiops), classified to Simian mastadenovirus F (SAdV-F). We identified unique features in the gene content of these SAdVs compared to those typical for other members of the genus Mastadenovirus. Namely, in the E1B region of SAdV-2, the 19K gene was replaced by an ITR repetition and a copy of the E4 ORF1 gene. Among the 37 genes in both SAdVs, three genes of different lengths, predicted to code for the cellular attachment proteins (the fibers), were found. These proteins exhibit high diversity. Yet, phylogenetic calculations of their conserved parts could reveal the probable evolutionary steps leading to the multiple-fibered contemporary HAdV and SAdV species. Seemingly, there existed (a) common ancestor(s) with two fiber genes for the lineages of the AdVs in species SAdV-B, -E, -F and HAdV-F, alongside a double-fibered ancestor for today's SAdV-C and HAdV-G, which later diverged into descendants forming today's species. Additionally, some HAdV-G members picked up a third fiber gene either to the left-hand or to the in-between position from the existing two. A SAdV-F progenitor also obtained a third copy to the middle, as observed in SAdV-17. The existence of three fiber genes in these contemporary AdVs brings novel possibilities for the design of optimised AdV-based vectors with potential multiple target binding abilities.

Разработка и характеристика векторной системы на основе аденовируса обезьян 25-го серотипа

Technological versatility and the humoral and cellular immune response induction capacity have conditioned wide spread of adenoviral vectors as vaccine and gene therapy drugs. However, vaccination with Sputnik V made a significant portion of the population immune to the types 5 and 26 (Ad5 and Ad26) recombinant human adenovirus vectors, which are some of the most frequently used bases for candidate vaccines. Today, vaccine designers tend to select alternative adenovirus serotypes as platforms to develop vaccines against new pathogens on. A good example is simian adenovirus type 25 (SAd25), which belongs to subgroup E. It is genetically distant from Ad5 and exhibits extremely low seroprevalence in human beings, which makes it an appealing alternative vaccine vector. The purpose of this work was to design and study a new vaccine platform based on simian adenovirus type 25. We relied on the advanced methods of molecular biology and virology to construct and make recombinant adenoviruses; the phylogenetic analysis in the context of this study was enabled with bioinformatic methods. The resulting recombinant adenoviral vector can effectively replicate itself in the HEK293 cell line (human embryonic kidney cells). This work substantiates the expediency of further investigation into the SAd25 vector as a platform for development of the prevention vaccines against various infectious diseases.

In vivo editing of the pan-endothelium by immunity evading simian adenoviral vector

Biological applications deriving from the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 site-specific nuclease system continue to impact and accelerate gene therapy strategies. Safe and effective in vivo co-delivery of the CRISPR/Cas9 system to target somatic cells is essential in the clinical therapeutic context. Both non-viral and viral vector systems have been applied for this delivery matter. Despite elegant proof-of-principle studies, available vector technologies still face challenges that restrict the application of CRISPR/Cas9-facilitated gene therapy. Of note, the mandated co-delivery of the gene-editing components must be accomplished in the potential presence of pre-formed anti-vector immunity. Additionally, methods must be sought to limit the potential of off-target editing. To this end, we have exploited the molecular promiscuities of adenovirus (Ad) to address the key requirements of CRISPR/Cas9-facilitated gene therapy. In this regard, we have endeavored capsid engineering of a simian (chimpanzee) adenovirus isolate 36 (SAd36) to achieve targeted modifications of vector tropism. The SAd36 vector with the myeloid cell-binding peptide (MBP) incorporated in the capsid has allowed selective in vivo modifications of the vascular endothelium. Importantly, vascular endothelium can serve as an effective non-hepatic cellular source of deficient serum factors relevant to several inherited genetic disorders. In addition to allowing for re-directed tropism, capsid engineering of nonhuman primate Ads provide the means to circumvent pre-formed vector immunity. Herein we have generated a SAd36. MBP vector that can serve as a single intravenously administered agent allowing effective and selective in vivo editing for endothelial target cells of the mouse spleen, brain and kidney. Data availabilityThe data that support the findings of this study are available from the corresponding author upon reasonable request.

Simian adenoviruses: Molecular and serological survey in monkeys and humans in Thailand

Simian adenoviruses are in the genus Mastadenovirus of the family Adenoviridae. This family is composed of non-enveloped, double-stranded DNA viruses that infect a wide range of animals. Mastadenoviruses infect mammals, including non-human primates and humans. The close genetic relatedness between simian and human adenoviruses, with its associated potential for the cross-species transmission of zoonotic adenoviruses from monkeys to humans and vice versa, poses important health concerns and thus warrants research. In this study, we performed a molecular survey of adenoviruses in monkeys in Thailand. Most of the monkeys tested here were long-tailed macaques, free-ranging in areas close to human territories across four provinces: Ratchaburi, Kanchanaburi, Lopburi, and Prachuap Khiri Khan. A few fecal samples from captive wild monkeys (a stump-tailed macaque, pig-tailed macaques, gibbons, and a leaf monkey) were also tested. Adenoviruses were detected in 33.3% (70 out of 210) of the fecal or rectal swab samples. The viruses identified in these samples included Simian adenovirus (SAdV)-A, SAdV-B, SAdV-H, Human adenovirus (HAdV)-D, HAdV-G, and a bat adenovirus species. One SAdV-B, SAdV RBR-7-10, was isolated from a long-tailed macaque fecal sample and identified by mass spectrometry. Its full hexon gene and nearly complete DNA polymerase gene were sequenced and analyzed, and the virions were imaged by transmission electron microscopy. The SAdV RBR-7-10 virus was used in a microneutralization assay to identify virus-specific antibodies in monkey plasma and human serum samples collected from the same areas in Prachuap Khiri Khan Province. We detected neutralizing antibodies against SAdV RBR-7-10 in 6.8% (n = 103) of the monkey samples but in none of the 125 human serum samples, suggesting no cross-species transmission of SAdV RBR-7-10 occurred at this study site. Nevertheless, a continuing surveillance of pathogens in monkeys is warranted to quickly identify possible emerging zoonotic outbreaks.

ChAdOx1 nCoV-19 (AZD1222) or nCoV-19-Beta (AZD2816) protect Syrian hamsters against Beta Delta and Omicron variants

ChAdOx1 nCoV-19 (AZD1222) is a replication-deficient simian adenovirus–vectored vaccine encoding the spike (S) protein of SARS-CoV-2, based on the first published full-length sequence (Wuhan-1). AZD1222 has been shown to have 74% vaccine efficacy against symptomatic disease in clinical trials. However, variants of concern (VoCs) have been detected, with substitutions that are associated with a reduction in virus neutralizing antibody titer. Updating vaccines to include S proteins of VoCs may be beneficial, even though current real-world data is suggesting good efficacy following boosting with vaccines encoding the ancestral S protein. Using the Syrian hamster model, we evaluate the effect of a single dose of AZD2816, encoding the S protein of the Beta VoC, and efficacy of AZD1222/AZD2816 as a heterologous primary series against challenge with the Beta or Delta variant. Minimal to no viral sgRNA could be detected in lungs of vaccinated animals obtained at 3- or 5- days post inoculation, in contrast to lungs of control animals. In Omicron-challenged hamsters, a single dose of AZD2816 or AZD1222 reduced virus shedding. Thus, these vaccination regimens are protective against the Beta, Delta, and Omicron VoCs in the hamster model.

Vaccines based on the replication-deficient simian adenoviral vector ChAdOx1: Standardized template with key considerations for a risk/benefit assessment

Replication-deficient adenoviral vectors have been under investigation as a platform technology for vaccine development for several years and have recently been successfully deployed as an effective COVID-19 counter measure. A replication-deficient adenoviral vector based on the simian adenovirus type Y25 and named ChAdOx1 has been evaluated in several clinical trials since 2012.The Brighton Collaboration Benefit-Risk Assessment of VAccines by TechnolOgy (BRAVATO) was formed to evaluate the safety and other key features of new platform technology vaccines. This manuscript reviews key features of the ChAdOx1-vectored vaccines.The simian adenovirus Y25 was chosen as a strategy to circumvent pre-existing immunity to common human adenovirus serotypes which could impair immune responses induced by adenoviral vectored vaccines. Deletion of the E1 gene renders the ChAdOx1 vector replication incompetent and further genetic engineering of the E3 and E4 genes allows for increased insertional capability and optimizes vaccine manufacturing processes. ChAdOx1 vectored vaccines can be manufactured in E1 complementing cell lines at scale and are thermostable. The first ChAdOx1 vectored vaccines approved for human use, against SARS-CoV-2, received emergency use authorization in the UK on 30th December 2020, and is now approved in more than 180 countries.Safety data were compiled from phase I-III clinical trials of ChAdOx1 vectored vaccines expressing different antigens (influenza, tuberculosis, malaria, meningococcal B, prostate cancer, MERS-CoV, Chikungunya, Zika and SARS-CoV-2), conducted by the University of Oxford, as well as post marketing surveillance data for the COVID-19 Oxford-AstraZeneca vaccine. Overall, ChAdOx1 vectored vaccines have been well tolerated. Very rarely, thrombosis with thrombocytopenia syndrome (TTS), capillary leak syndrome (CLS), immune thrombocytopenia (ITP), and Guillain-Barre syndrome (GBS) have been reported following mass administration of the COVID-19 Oxford-AstraZeneca vaccine. The benefits of this COVID-19 vaccination have outweighed the risks of serious adverse events in most settings, especially with mitigation of risks when possible.Extensive immunogenicity clinical evaluation of ChAdOx1 vectored vaccines reveal strong, durable humoral and cellular immune responses to date; studies to refine the COVID-19 protection (e.g., via homologous/heterologous booster, fractional dose) are also underway.New prophylactic and therapeutic vaccines based on the ChAdOx1 vector are currently undergoing pre-clinical and clinical assessment, including vaccines against viral hemorrhagic fevers, Nipah virus, HIV, Hepatitis B, amongst others.

A pan-genotype hepatitis C virus viral vector vaccine generates T cells and neutralizing antibodies in mice.

A prophylactic vaccine targeting multiple HCV genotypes (gt) is urgently required to meet World Health Organization elimination targets. Neutralizing antibodies (nAbs) and CD4+ and CD8+ T cells are associated with spontaneous clearance of HCV, and each may contribute to protective immunity. However, current vaccine candidates generate either nAbs or T cells targeting genetically variable epitopes and have failed to show efficacy in human trials. We have previously shown that a simian adenovirus vector (ChAdOx1) encoding conserved sequences across gt1-6 (ChAd-Gt1-6), and separately gt-1a E2 protein with variable regions deleted (E2Δ123HMW ), generates pan-genotypic T cells and nAbs, respectively. We now aim to develop a vaccine to generate both viral-specific B- and T-cell responses concurrently. We show that vaccinating with ChAd-Gt1-6 and E2Δ123HMW sequentially in mice generates T-cell and antibody (Ab) responses comparable to either vaccine given alone. We encoded E2Δ123 in ChAdOx1 (ChAd-E2Δ123) and show that this, given with an E2Δ123HMW protein boost, induces greater CD81-E2 inhibitory and HCV-pseudoparticle nAb titers compared to the E2Δ123HMW prime boost. We developed bivalent viral vector vaccines (ChAdOx1 and modified vaccinia Ankara [MVA]) encoding both Gt1-6 and E2Δ123 immunogens (Gt1-6-E2Δ123) generating polyfunctional CD4+ and CD8+ T cells and nAb titers in prime/boost strategies. This approach generated nAb responses comparable to monovalent E2Δ123 ChAd/MVA vaccines and superior to three doses of recombinant E2Δ123HMW protein, while also generating high-magnitude T-cell responses. These data are an important step forward for the development of a pan-genotype HCV vaccine to elicit T cells and nAbs for future assessment in humans.

Development of Viral-Vectored Vaccines and Virus Replicon Particle-Based Neutralisation Assay against Mayaro Virus.

Mayaro virus (MAYV) is an emerging alphavirus causing acute febrile illness associated with chronic polyarthralgia. Although MAYV is currently restricted to tropical regions in South America around the Amazon basin, it has the potential to spread globally by Aedes species mosquitoes. In addition, there are currently no specific therapeutics or licenced vaccines against MAYV infection. We have previously shown that an adenovirus based Mayaro vaccine (ChAdOx1 May) was able to provide full protection against MAYV challenge in vaccinated A129 mice and induced high neutralising antibody titres. In this study, we have constructed a replication deficient simian adenovirus (ChAdOx2) and a Modified Ankara Virus (MVA) based vaccine expressing the MAYV structural cassette (sMAYV) similar to ChAdOx1 May, and characterised recombinant MAYV E2 glycoprotein expressed in a mammalian system for immune monitoring. We demonstrate that ChAdOx2 May was able to induce high antibody titres similar to ChAdOx1 May, and MVA May was shown to be an effective boosting strategy following prime vaccination with ChAdOx1 or ChAdOx2 May. In order to measure MAYV neutralising ability, we have developed a virus replicon particle-based neutralisation assay which effectively detected neutralising antibodies against MAYV. In summary, our study indicates the potential for further clinical development of the viral vectored MAYV vaccines against MAYV infections.

Restriction-Assembly: A Solution to Construct Novel Adenovirus Vector.

Gene therapy and vaccine development need more novel adenovirus vectors. Here, we attempt to provide strategies to construct adenovirus vectors based on restriction-assembly for researchers with little experience in this field. Restriction-assembly is a combined method of restriction digestion and Gibson assembly, by which the major part of the obtained plasmid comes from digested DNA fragments instead of PCR products. We demonstrated the capability of restriction-assembly in manipulating the genome of simian adenovirus 1 (SAdV-1) in this study. A PCR product of the plasmid backbone was combined with SAdV-1 genomic DNA to construct an infectious clone, plasmid pKSAV1, by Gibson assembly. Restriction-assembly was performed repeatedly in the steps of intermediate plasmid isolation, modification, and restoration. The generated adenoviral plasmid was linearized by restriction enzyme digestion and transfected into packaging 293 cells to rescue E3-deleted replication-competent SAdV1XE3-CGA virus. Interestingly, SAdV1XE3-CGA could propagate in human chronic myelogenous leukemia K562 cells. The E1 region was similarly modified to generate E1/E3-deleted replication-defective virus SAdV1-EG. SAdV1-EG had a moderate gene transfer ability to adherent mammalian cells, and it could efficiently transduce suspension cells when compared with the human adenovirus 5 control vector. Restriction-assembly is easy to use and can be performed without special experimental materials and instruments. It is highly effective with verifiable outcomes at each step. More importantly, restriction-assembly makes the established vector system modifiable, upgradable and under sustainable development, and it can serve as the instructive method or strategy for the synthetic biology of adenoviruses.