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Characterization of a Family of Chimpanzee Adenoviruses and Development of Molecular Clones for Gene Transfer Vectors
Abstract
The high prevalence of preexisting immunity to the commonly used adenoviral vectors, as well as the requirement for readministration of vector for multiple therapeutic applications, necessitates the development of a panel of immunologically distinct adenoviral vectors against which neutralizing antibodies are rare in human populations. We have completely sequenced three chimpanzee-derived adenoviruses, Pan 5, Pan 6, and Pan 7, and have molecularly cloned E1-deleted vector genomes from each as bacterial plasmids. All the E1-deleted vectors were grown to high titer in HEK 293 cells. Neutralizing antibodies to the chimpanzee adenoviral vectors were not detected in serum samples from human subjects. In vitro cross-neutralization using rabbit antisera and in vivo readministration experiments in mice demonstrated that antibodies against Pan 5, Pan 7, or Pan 9 cross-neutralize one another but do not neutralize Pan 6. These results indicate that chimpanzee adenoviral vectors may be useful as vaccines or gene therapy vectors in human populations and should allow applications that require multiple vector administrations.
... administration; in this preparation, IV-Ig had an Ad5 NAb titer of 1/2,560. Polyclonal rabbit serum against a serologically distinct simian Ad (SAdV-24 9 ) failed to enhance transduction of Ad5-GFP on human DCs (mean fluorescence intensity [MFI] = 8.5). Our studies are consistent with others that reported Ab-enhanced transduction of adenoviral vectors on DCs. ...
... Polyclonal serum against Ad5 was generated by repeatedly immunizing rabbits with the respective vectors. 9 In brief, New Zealand white rabbits received intramuscular injections of 10 13 vp of recombinant Ad5.CMV.GFP, followed by a boost with an equal dose with Freund's incomplete adjuvant 34 days later. The animals were terminally bled 14 days after the boost to collect antiserum that had an NAb titer of 81,920 (reciprocal of serum dilution). ...
... Polyclonal rabbit serum against SAdV-24, a chimpanzee-derived adenoviral vector, does not cross-react with Ad5. 9 Serum from human subjects pre-and day 28 post-Ad5 vector administration was kindly provided by Dr. David Margolis (Department of Dermatology, University of Pennsylvania). The samples were obtained from subjects enrolled in a phase I clinical trial (ClinicalTrials.gov: ...
Intra-arterial administration of an adenovirus serotype 5 (Ad5) vector in a gene therapy trial caused lethal, systemic inflammation in subject 019 with ornithine transcarbamylase deficiency. This unanticipated inflammatory response was absent in another subject receiving the same vector dose and in 16 subjects receiving lower vector doses. We hypothesized that an immune memory to a previous natural adenovirus infection enhanced the immune response to high-dose systemic Ad5 vector, causing the exaggerated immune response in subject 019. To investigate this, we found that rabbit polyclonal sera to Ad5 and pooled human immunoglobulin (Ig) inhibited Ad5 vector transduction of non-immune cells in vitro, but enhanced transduction and activation of human dendritic cells (DCs). Sera from approximately 7% of normal human subjects and 50% of patients treated topically with Ad5 vectors enhanced Ad5 transduction and activation of DCs, apparently from formation of Ig-Ad5 immune complexes and binding to DCs through FcγR. Subject 019's blood substantially increased Ad5-vector activation of human DC primary cultures at levels exceeding those from normal subjects. Although this study is based on one event in a single subject, the results implicate a pre-existing humoral immune response to Ad5 in the lethal systemic inflammatory response that occurred in subject 019.
... Immunogenicity and protective activity of AdC68-S were systematically evaluated against lethal challenge with a mouse-adapted MERS-CoV (MERS-CoV-MA) in our previously developed human DPP4 knock-in (hDPP4-KI) mouse model [39]. Of the chimpanzee adenoviruses, we chose AdC68 as a vector due to its low pre-existing immunity and overall safety profile [40][41][42]. AdC68 has only 0-2% seropositivity in humans, compared with human adenovirus serotype 5 (HuAd5) which has 75-80% seroprevalence [41]. Recombinant vaccines based on AdC68 and other rare serotype chimpanzee adenoviral vectors, such as ChAd63 and ChAd3, have recently been engineered to express various antigens and some have demonstrated impressive safety and immunogenicity profiles in clinical studies [43][44][45]. ...
... There are two unique aspects of our study can be highlighted here. The first is related to the AdC68 vector, which has preferred profiles of low pre-existing immunity and overall safety in animals and humans [40][41][42]. This is clearly advantageous over many human adenoviral vectors that are widely used in vaccine development, such as human adenovirus 5 (HuAd5). ...
... Furthermore, both PanAd3-RSV, which encodes the RSV proteins F, N, and M2-1, and ChAd3-NSmut, which encodes the hepatitis C virus proteins NS3, NS4, NS5A, and NS5B, elicited strong immune responses in clinical trial participants [44]. These results demonstrate that chimpanzee adenoviral vectors have impressive safety, tolerability, and immunogenicity profiles in humans and highlight their promise for vaccine development [40,41]. ...
The recently identified Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe and fatal acute respiratory illness in humans. However, no approved prophylactic and therapeutic interventions are currently available. The MERS-CoV envelope spike protein serves as a crucial target for neutralizing antibodies and vaccine development, as it plays a critical role in mediating viral entry through interactions with the cellular receptor, dipeptidyl peptidase 4 (DPP4). Here, we constructed a recombinant rare serotype of the chimpanzee adenovirus 68 (AdC68) that expresses full-length MERS-CoV S protein (AdC68-S). Single intranasal immunization with AdC68-S induced robust and sustained neutralizing antibody and T cell responses in BALB/c mice. In a human DPP4 knock-in (hDPP4-KI) mouse model, it completely protected against lethal challenge with a mouse-adapted MERS-CoV (MERS-CoV-MA). Passive transfer of immune sera to naïve hDPP4-KI mice also provided survival advantages from lethal MERS-CoV-MA challenge. Analysis of sera absorption and isolated monoclonal antibodies from immunized mice demonstrated that the potent and broad neutralizing activity was largely attributed to antibodies targeting the receptor binding domain (RBD) of the S protein. These results show that AdC68-S can induce protective immune responses in mice and represent a promising candidate for further development against MERS-CoV infection in both dromedaries and humans.
... An additional observation is that upon re-administration liver toxicity is increased [90], and in human blood neutralizing antibodies may activate complement and induce inflammatory reactions [91]. In an attempt to circumvent this issue Ad vectors derived from different human [83,84,[92][93][94][95][96][97][98][99][100][101][102][103][104] and animal [105][106][107][108][109][110] serotypes, to which the human population has a lower prevalence of neutralizing antibodies, are currently being investigated. Non-replicating versions of alternate serotypes face similar issues to the first-, second generation and HC-Ads based on Ad5 serotype, regarding which producer cell line is suitable to achieve production and propagation of the vector. ...
... Several non-human serotypes derived from bovine [106], canine [105,107] and simian [108][109][110]117] sources have been developed as potential E1-deleted Ad vectors for use as gene delivery or vaccine vectors for human therapies. In the case of the bovine, BAV-3 and canine, CAV-2 vectors, specialized E1 producer cells based on appropriate species cell lines, such as bovine kidney and fetal retinal cells expressing Ad5 E1 sequences [106] and canine kidney cells expressing the CAV-2 E1 [105], have been developed to package and propagate these vectors. ...
... In the case of the bovine, BAV-3 and canine, CAV-2 vectors, specialized E1 producer cells based on appropriate species cell lines, such as bovine kidney and fetal retinal cells expressing Ad5 E1 sequences [106] and canine kidney cells expressing the CAV-2 E1 [105], have been developed to package and propagate these vectors. The chimpanzee derived Ad vectors of subgroup E can be propagated on HEK293 cells and therefore can be grown under standard methods [108,109]. However, as with Ad11, Ad35 and other human subgroup B vectors, chimpanzee derived Ad vectors of subgroup B cannot be propagated on regular E1 producer cell lines. ...
Adenovirus (Ad) vectors, in particular those of the serotype 5, are highly attractive for a wide range of gene therapy, vaccine and virotherapy applications (as discussed in further detail in this issue). Wild type Ad5 virus can replicate in numerous tissue types but to use Ad vectors for therapeutic purposes the viral genome requires modification. In particular, if the viral genome is modified in such a way that the viral life cycle is interfered with, a specific producer cell line is required to provide trans-complementation to overcome the modification and allow viral production. This can occur in two ways; use of a producer cell line that contains specific adenoviral sequences incorporated into the cell genome to trans-complement, or use of a producer cell line that naturally complements for the modified Ad vector genome. This review concentrates on producer cell lines that complement non-replicating adenoviral vectors, starting with the historical HEK293 cell line developed in 1977 for first generation Ad vectors. In addition the problem of replication-competent adenovirus (RCA) contamination in viral preparations from HEK293 cells is addressed leading to the development of alternate cell lines. Furthermore novel cell lines for more complex Ad vectors and alternate serotype Ad vectors are discussed.
... Subsequently, the release into GenBank of 16 additional genome data sets from the authors, HAdV-B3 (two genomes), HAdV-B7 (three genomes), HAdV-B16, HAdV-B21, HAdV-B50, HAdV-B14, HAdV-B34, HAdV-C5, HAdV-C6 (embargoed) and HAdV-E4 (four genomes), added to the growing number of available HAdV genomes [25,[56][57][58][59][60]. Also available were five chimpanzee AdV genomes [61,62], which are of interest as alternative gene delivery vectors. Currently, there are 31 prototype and 37 field isolate genomes deposited in GenBank, with the number expected to grow as clinical investigations of HAdV-associated illnesses are leading to the identification of putatively interesting HAdVs. ...
... One review by Stone et al. [81] is an example. Primary literature citations include [2,4,7,53,62,[82][83][84][85][86][87][88][89]. It is anticipated that genomics and bioinformatics resources will aid in these on-going work and development. ...
... A recent contribution of 33 novel non-human primate genomes (30 ape and three macaque) has been reported and deposited in GenBank [93]. These are in addition to several monkey AdV genomes, sporadically deposited as simian AdV (SAdV) since 2004, and the five original chimpanzee AdV genomes, also noted as SAdVs, deposited into GenBank in 2004 [61,62]. The first chimpanzee AdVs were originally deposited at the American Type Culture Collection (ATCC) in the 1960s-70s, so these 33 additional genomes represent a recent, renewed and directed interest in novel non-human primate AdVs. ...
Technological advances and increasingly cost-effect methodologies in DNA sequencing and computational analysis are providing genome and proteome data for human adenovirus research. Applying these tools, data and derived knowledge to the development of vaccines against these pathogens will provide effective prophylactics. The same data and approaches can be applied to vector development for gene delivery in gene therapy and vaccine delivery protocols. Examination of several field strain genomes and their analyses provide examples of data that are available using these approaches. An example of the development of HAdV-B3 both as a vaccine and also as a vector is presented.
... We chose AdC68 as the backbone to express our influenza-SARS-CoV-2 doubly-targeting immunogen because of its low preexisting immunity and overall safety profile (11,12). Compared with human adenovirus serotype 5 (HuAd5), which has 75 to 80% seroprevalence, AdC68 has only 0 to 2% seropositivity in humans (13). ...
... The shift in size caused by deglycosylation was consistent with the presence of two N-glycosylation sites (N331 and N343) in the RBD, as previously reported (14). Given that an individual N-glycosylation site is estimated to contribute Purified BSA and RBD-ferritin proteins were treated or untreated with a deglycosylation mix under native conditions (1) versus more effectively denaturing conditions (11) and were then subjected to SDS gel electrophoresis. The separated proteins were visualized by Coomassie blue staining. ...
The ongoing SARS-CoV-2 pandemic posed a severe global threat on public health, as do so by influenza viruses (influenza) and other coronaviruses. Here we present chimpanzee adenovirus 68 (AdC68)-based vaccines designed to universally target coronaviruses and influenza. Our design is centered on an immunogen generated by fusing the SARS-CoV-2 receptor-binding domain (RBD) to the conserved stalk of H7N9 hemagglutinin (HA). Remarkably, the constructed vaccine effectively induced both SARS-CoV-2-targeting antibodies and anti-influenza antibodies in mice, consequently affording protection from lethal SARS-CoV-2 and H7N9 challenges and effective H3N2 control. We propose our AdC68 vectored coronavirus-influenza vaccine as a universal approach toward curbing respiratory virus-causing pandemics.
IMPORTANCE
The COVID-19 pandemic exemplifies the severe public health threat of respiratory virus infection, as do so by influenza A viruses. The currently envisioned strategy for prevention of respiratory virus-causing diseases requires comprehensive administration of vaccines tailored for individual virus. Here we present an alternative strategy by designing chimpanzee adenovirus 68-based vaccines targeting both SARS-CoV-2 receptor-binding-domain and conserved stalk of influenza hemagglutinin. When tested in mice, this strategy attained potent neutralizing antibodies against wild-type SARS-CoV-2 as well as its emerging variants, enabling an effective protection against lethal SARS-CoV-2 challenge. Notably, it also entitled a complete protection from lethal H7N9 challenge and efficient control of H3N2-induced morbidity. Our study opens a new avenue to universally curb respiratory virus infection by vaccination.
... evaluated by a Human Adenovirus Working Group. Rather than being simply a basic biological research interest, SAdVs are increasingly recognized as important to human health from both an epidemiological perspective, e.g., zoonosis (Purkayastha et al., 2005a;Purkayastha et al., 2005b;Dehghan et al., 2013b), and a clinical applications perspective, e.g., gene delivery vectors (Graham and Prevec, 1992;Roy et al., 2004;Roy et al., 2006) and as oncolytic agents (Doronin and Shayakhmetov, 2012;Larson et al., 2015). Sequence recombination within the separate groups of HAdV and SAdV genomes has been reported as an important mechanism of adenovirus evolution, leading to novel and emergent viruses and pathogens (Walsh et al., 2009;Walsh et al., 2010;Robinson et al., 2011;Robinson et al., 2013;Hoppe et al., 2015b). ...
... To provide a high-resolution and more comprehensive view of the phylogenetic relationships and taxonomy of HAdVs and SAdVs, this report uses a broader examination of all published HAdV and SAdV genomes to yield heuristically parsimonious phylogenomic trees. This analysis yields a revised, broad taxonomic view of an intertwined phylogenetic tree with implications for human health, not only because these viruses are putative emergent and re-emergent pathogens (Xiang et al., 2006;Roy et al., 2009;Ersching et al., 2010;Wevers et al., 2011;Chen et al., 2011b;Chiu et al., 2013;Pauly et al., 2015;Hoppe et al., 2015a), but also because of their use as potential gene delivery and gene therapy vectors for medical therapies (Graham and Prevec, 1992;Roy et al., 2004;Roy et al., 2006). The latter is important as SAdVs have been presumed to be attractive, alternative vectors that bypass pre-existing human prior exposure and resultant immunity. ...
With the advent of high‐resolution and cost‐effective genomics and bioinformatics tools and methods contributing to a large database of both human (HAdV) and simian (SAdV) adenoviruses, a genomics‐based re‐evaluation of their taxonomy is warranted. Interest in these particular adenoviruses is growing in part due to the applications of both in gene transfer protocols, including gene therapy and vaccines, as well in oncolytic protocols. In particular, the re‐evaluation of SAdVs as appropriate vectors in humans is important as zoonosis precludes the assumption that human immune system may be naïve to these vectors. Additionally, as important pathogens, adenoviruses are a model organism system for understanding viral pathogen emergence through zoonosis and anthroponosis, particularly among the primate species, along with recombination, host adaptation, and selection, as evidenced by one long‐standing human respiratory pathogen HAdV‐4 and a recent re‐evaluation of another, HAdV‐76. The latter reflects the insights on amphizoonosis, defined as infections in both directions among host species including “other than human”, that are possible with the growing database of nonhuman adenovirus genomes. HAdV‐76 is a recombinant that has been isolated from human, chimpanzee, and bonobo hosts. On‐going and potential impacts of adenoviruses on public health and translational medicine drive this evaluation of 174 whole genome sequences from HAdVs and SAdVs archived in GenBank. The conclusion is that rather than separate HAdV and SAdV phylogenetic lineages, a single, intertwined tree is observed with all HAdVs and SAdVs forming mixed clades. Therefore, a single designation of “primate adenovirus” (PrAdV) superseding either HAdV and SAdV is proposed, or alternatively, keeping HAdV for human adenovirus but expanding the SAdV nomenclature officially to include host species identification as in ChAdV for chimpanzee adenovirus, GoAdV for gorilla adenovirus, BoAdV for bonobo adenovirus, and ad libitum.
... Viral vaccine vectors included modified vaccinia virus Ankara (MVA), human adenovirus serotype 5 (AdHu5), and the simian adenoviruses (SAd) serotypes 6 (C6), 7 (C7), 9 (C9), and 63 (AdCh63). PfM115 was cloned into SAd viral vectors C6, C7, and C9 using methods described previously (48). Insertion of PfM115 and PfM128 into AdHu5 and MVA and of PfM128 into AdCh63 was performed using methods previously described for other antigen inserts (13,44). ...
... Where PfM128 was expressed as the antigen, this is additionally denoted (e.g., AdHu5_M PfM128). C9 has been demonstrated to cross-react with C7 neutralizing antibodies and was therefore not used as a boosting agent (48). Overlapping peptide pools were used to map T cell epitopes from PfM115 in mice (see Fig. S2 in the supplemental material). ...
Although merozoite surface protein 1 (MSP-1) is a leading candidate vaccine antigen for blood-stage malaria, its efficacy
in clinical trials has been limited in part by antigenic polymorphism and potentially by the inability of protein-in-adjuvant
vaccines to induce strong cellular immunity. Here we report the design of novel vectored Plasmodium falciparum vaccines capable of overcoming such limitations. We optimized an antigenic insert comprising the four conserved blocks of
MSP-1 fused to tandemly arranged sequences that represent both allelic forms of the dimorphic 42-kDa C-terminal region. Inserts
were expressed by adenoviral and poxviral vectors and employed in heterologous prime-boost regimens. Simian adenoviral vectors
were used in an effort to circumvent preexisting immunity to human adenoviruses. In preclinical studies these vaccines induced
potent cellular immune responses and high-titer antibodies directed against MSP-1. The antibodies induced were found to have
growth-inhibitory activity against dimorphic allelic families of P. falciparum. These vectored vaccines should allow assessment in humans of the safety and efficacy of inducing strong cellular as well
as cross-strain humoral immunity to P. falciparum MSP-1.
... Additionally, several adenoviruses infecting other mammals and capable of infecting human cells have been investigated, including adenovirus from bovine type 3 (Mittal et al., 1995), chimpanzee (ChAd) type 68 (Xiang et al., 2002), types 5, 6, 7 (Roy et al., 2004), C1 (Tatsis et al., 2007a) and Y25 (Dicks et al., 2012), rhesus monkey types 51, 52 and 53 (Abbink et al., 2015), porcine type 3 (Bangari and Mittal, 2004) and simian type 21 (Roy et al., 2006). Clinical trial data indicated that ChAd63 is safe and induces a strong immune response (O'Hara et al., 2012). ...
Adenovirus was first identified in the 1950s and since then this pathogenic group of viruses has been explored and transformed into a genetic transfer vehicle. Modification or deletion of few genes are necessary to transform it into a conditionally or non-replicative vector, creating a versatile tool capable of transducing different tissues and inducing high levels of transgene expression. In the early years of vector development, the application in monogenic diseases faced several hurdles, including short-term gene expression and even a fatality. On the other hand, an adenoviral delivery strategy for treatment of cancer was the first approved gene therapy product. There is an increasing interest in expressing transgenes with therapeutic potential targeting the cancer hallmarks, inhibiting metastasis, inducing cancer cell death or modulating the immune system to attack the tumor cells. Replicative adenovirus as vaccines may be even older and date to a few years of its discovery, application of non-replicative adenovirus for vaccination against different microorganisms has been investigated, but only recently, it demonstrated its full potential being one of the leading vaccination tools for COVID-19. This is not a new vector nor a new technology, but the result of decades of careful and intense work in this field.
... Several simian Adenovirus have been employed as E1-deleted Ad vectors for gene delivery or vaccine vectors exploited in human therapies [36][37][38][39]. Chimpanzee Ad vectors of E species belonging to Human Mastadenovirus E species (e.g., chAd63, ChAdOx-1) can be propagated on HEK293 cells, even if suboptimally [36]. ...
Sleeping Beauty (SB) is the first DNA transposon employed for efficient transposition in vertebrate cells, opening new applications for genetic engineering and gene therapies. A transposon-based gene delivery system holds the favourable features of non-viral vectors and an attractive safety profile. Here, we employed SB to engineer HEK293 cells for optimizing the production of a chimpanzee Adenovector (chAd) belonging to the Human Mastadenovirus C species. To date, chAd vectors are employed in several clinical settings for infectious diseases, last but not least COVID-19. A robust, efficient and quick viral vector production could advance the clinical application of chAd vectors. To this aim, we firstly swapped the hAd5 E1 with chAd-C E1 gene by using the CRISPR/Cas9 system. We demonstrated that in the absence of human Ad5 E1, chimp Ad-C E1 gene did not support HEK293 survival. To improve chAd-C vector production, we engineered HEK293 cells to stably express the chAd-C precursor terminal protein (ch.pTP), which plays a crucial role in chimpanzee Adenoviral DNA replication. The results indicate that exogenous ch.pTP expression significantly ameliorate the packaging and amplification of recombinant chAd-C vectors thus, the engineered HEK293ch.pTP cells could represent a superior packaging cell line for the production of these vectors.
... A negative staining EM investigation showed the typical patterns (Sutjipto et al., 2005) seen for empty and full/ mature capsids, respectively (data not shown). Tests for infectivity ( Table 2) showed that the particles from the lowdensity fraction exhibited extremely low infectivity as expected (Burlingham et al., 1974;Daniell, 1976) while the particles from the high-density fraction displayed infectivity ratios in line with expectations for the product and recombinant chimpanzee adenovirus (Roy et al., 2004). DCS analysis ( Figure 2B, C) revealed that the particles from the low-density fraction aligned with the previously labeled empty capsid region and that the particles from the high-density fraction contained both the previously labeled intermediate and full capsid region. ...
We observed differential infectivity and product yield between two recombinant chimpanzee adenovirus C68 constructs whose primary difference was genome length. To determine a possible reason for this outcome, we characterized the proportion and composition of the empty and packaged capsids. Both analytical ultracentrifugation (AUC) and differential centrifugation sedimentation (DCS, a rapid and quantitative method for measuring adenoviral packaging variants) were employed for an initial assessment of genome packaging and showed multiple species whose abundance deviated between the virus builds but not manufacturing campaigns. Identity of the packaging variants was confirmed by charge detection mass spectrometry (CDMS), the first known application of this technique to analyze adenovirus. The empty and packaged capsid populations were separated via preparative ultracentrifugation and then combined into a series of mixtures. These mixtures showed the oft-utilized denaturing A260 adenoviral particle titer method will underestimate the actual particle titer by as much as three-fold depending on the empty/full ratio. In contrast, liquid chromatography with fluorescence detection proves to be a superior viral particle titer methodology.
... Most of the ChAd vectors are generated by replacing the E1 region of the genome with a transgene. In addition, to overcome safety issues and/or increase transgene insertion capacity, E3 and/or E4 genes are also deleted [33,34]. Other changes were also explored to improve in vitro productivity by manipulating small non-coding RNAs [35]. ...
Several human adenoviral (Ad) vectors have been developed for vaccine delivery owing to their numerous advantages, including the feasibility of different vector designs, the robustness of elicited immune responses, safety, and scalability. To expand the repertoire of Ad vectors for receptor usage and circumvention of Ad vector immunity, the use of less prevalent human Ad types or nonhuman Ads were explored for vector design. Notably, many nonhuman Ad vectors have shown great promise in preclinical and clinical studies as vectors for vaccine delivery. This review describes the key features of several nonhuman Ad vector platforms and their implications in developing effective vaccines against infectious diseases.
... For comparison, the frequency of the sua gene in S. parauberis was 27.3%, which agreed with results of previous studies (Luther et al. 2008;Reinoso et al. 2011). Plasminogen activators have long been thought to be involved in the colonization and growth of streptococci and to be capable of transforming plasminogen into plasmin; along with the ability to degrade extracellular matrix proteins, this results in an advantageous environment for colonization of pathogenic bacteria (Roy et al. 2004;Perrig et al. 2015). The plasminogen activator/ streptokinase-encoding gene pauA/skc is one of the most important virulence-associated invasiveness factors (Khan et al. 2003). ...
The objectives of this study were to investigate antimicrobial resistance of Streptococcus uberis and Streptococcus parauberis isolated from cows with bovine clinical mastitis in China and to examine the distribution of resistance- and virulence-related gene patterns. Antimicrobial susceptibility was determined by the E-test. Genes encoding antimicrobial resistance and invasiveness factors were examined by PCR. A total of 27 strains were obtained from 326 mastitis milk samples. Streptococcus parauberis isolates (n=11) showed high resistance to erythromycin (90.9%), followed by tetracycline (45.5%), chloramphenicol (36.4%) and clindamycin (27.3%). Streptococcus uberis isolates (n=16) were highly resistant to tetracycline (81.3%) and clindamycin (62.5%). Both species were susceptible to ampicillin. The most prevalent resistance gene in S. uberis was tetM (80.0%), followed by blaZ (62.5%) and ermB (62.5%). However, tetM, blaZ, and ermB genes were only found in 27.3, 45.5, and 27.3%, respectively, of S. parauberis. In addition, all of the isolates carried at least one selected virulence-related gene. The most prevalent virulence-associated gene pattern in the current study was sua+pauA/skc+gapC+hasC detected in 22.2% of the strains. One S. uberis strain carried 7 virulence-associated genes and belonged to the sua+pauA/skc+gapC+cfu+hasA+hasB+hasC pattern. More than 59.3% of analysed strains carried 4 to 7 virulence-related genes. Our findings demonstrated that S. parauberis and S. uberis isolated from clinical bovine mastitis cases in China exhibited diverse molecular ecology, and that the strains were highly resistant to antibiotics commonly used in the dairy cow industry. The data obtained in the current study contribute to a better understanding of the pathogenesis of bacteria in mastitis caused by these pathogens, and the findings are relevant to the development of multivalent vaccines and targeted prevention procedures.
... The virion exterior is composed of three major structural proteins, the fiber, the penton base and the hexon [ (15)(16)(17)(18); Figure 1]. Recombinant Ad (rAd) vectors can easily be rendered replication-incompetent (non-replicating) through deletion of the essential viral gene E1 from their genome and can be vectorized for easy manipulation (16,(19)(20)(21). Further improvements to these first-generation Ad vectors have been developed in which the E3 region is also deleted, to accommodate a larger heterologous transgene capacity of ∼7.5 kbp. ...
Adenoviral vectors are a safe and potently immunogenic vaccine delivery platform. Non-replicating Ad vectors possess several attributes which make them attractive vaccines for infectious disease, including their capacity for high titer growth, ease of manipulation, safety, and immunogenicity in clinical studies, as well as their compatibility with clinical manufacturing and thermo-stabilization procedures. In general, Ad vectors are immunogenic vaccines, which elicit robust transgene antigen-specific cellular (namely CD8⁺ T cells) and/or humoral immune responses. A large number of adenoviruses isolated from humans and non-human primates, which have low seroprevalence in humans, have been vectorized and tested as vaccines in animal models and humans. However, a distinct hierarchy of immunological potency has been identified between diverse Ad vectors, which unfortunately limits the potential use of many vectors which have otherwise desirable manufacturing characteristics. The precise mechanistic factors which underlie the profound disparities in immunogenicity are not clearly defined and are the subject of ongoing, detailed investigation. It has been suggested that a combination of factors contribute to the potent immunogenicity of particular Ad vectors, including the magnitude and duration of vaccine antigen expression following immunization. Furthermore, the excessive induction of Type I interferons by some Ad vectors has been suggested to impair transgene expression levels, dampening subsequent immune responses. Therefore, the induction of balanced, but not excessive stimulation of innate signaling is optimal. Entry factor binding or receptor usage of distinct Ad vectors can also affect their in vivo tropism following administration by different routes. The abundance and accessibility of innate immune cells and/or antigen-presenting cells at the site of injection contributes to early innate immune responses to Ad vaccination, affecting the outcome of the adaptive immune response. Although a significant amount of information exists regarding the tropism determinants of the common human adenovirus type-5 vector, very little is known about the receptor usage and tropism of rare species or non-human Ad vectors. Increased understanding of how different facets of the host response to Ad vectors contribute to their immunological potency will be essential for the development of optimized and customized Ad vaccine platforms for specific diseases.
... In addition to increased attention to HAdVs due to infection-associated morbidity, the biochemical and clinical characteristics of the virus, including its stability and ability to induce innate and adaptive immune responses in mammals, have made AdVs particularly attractive vectors for vaccines and gene therapy. The use of non-human simian AdVs (SAdVs) [22] to circumvent prior exposure associated immunity in humans is of importance. A recent review of HAdV seroprevalence studies reports that by 2018 there were hundreds of vaccine, gene therapy, and cancer trials using HAdV-based vectors [23]. ...
Adenoviruses (AdVs) are major contributors to clinical illnesses. Novel human and animal AdVs continue to be identified and characterized. Comparative analyses using bioinformatic methods and Omics-based technologies allow insights into how these human pathogens have emerged and their potential for host cross-species transmission. Systematic review of literature published across ProQuest, Pubmed, and Web of Science databases for evidence of adenoviral zoonotic potential identified 589 citations. After removing duplicates, 327 citations were screened for relevance; of which, 74 articles received full-text reviews. Among these, 24 were included here, of which 16 demonstrated evidence of zoonotic transmission of AdVs. These documented instances of AdV crossing host species barriers between humans and non-human primate, bat, feline, swine, canine, ovine, and caprine. Eight studies sought to but did not find evidence of zoonosis. The findings demonstrate substantial evidence suggesting AdVs have previously and will continue crossing host species barriers. These have human health consequences both in terms of novel pathogen emergence and epidemic outbreaks, and of appropriate and safe use of non-human adenoviruses for therapeutics. As routine human clinical diagnostics may miss a novel cross-species adenovirus infection in humans, next generation sequencing or panspecies molecular diagnostics may be necessary to detect such incursions.
... The recognition of zoonosis, anthroponosis, and recombination as key components and contributors of HAdV and SAdV genome and viral diversity suggests caution in the use of SAdV genomes as alternative vectors for epitope and gene delivery in vaccine development and gene therapy protocols (53). The hexon and fiber proteins, which are principal sites of antigenic epitopes of HAdV-B76, SAdV-B35.1, ...
Genomics analysis of a historically-intriguing and predicted emergent human adenovirus (HAdV) pathogen, which caused pneumonia and death, provides insight into a novel molecular evolution pathway involving “ping-pong” zoonosis and anthroponosis. The genome of this promiscuous pathogen is embedded with evidence of unprecedented multiple, multidirectional, stable, and reciprocal cross-species infections of hosts from three species (human, chimpanzee, and bonobo). This recombinant genome, typed as HAdV-B76, is identical to two recently reported simian AdV (SAdV) genomes isolated from chimpanzees and bonobos. Additionally, the presence of a critical adenoviral replication element found in HAdV genomes, in addition to genes that are highly similar with counterparts in other HAdVs, reinforce its potential as a human pathogen. Reservoirs in nonhuman hosts may explain periods of apparent absence and then re-emergence of human adenoviral pathogens, as well as present pathways for the genesis of those thought to be newly emergent. The nature of the HAdV-D76 genome has implications for the use of SAdVs as gene delivery vectors in human gene therapy and vaccines, selected to avoid preexisting and potentially fatal host immune responses to HAdV.
Importance: An emergent adenoviral human pathogen, HAdV-B76, associated with a fatality in 1965, shows a remarkable degree of genome identity with two recently isolated simian adenoviruses that contain cross-species genome recombination events from three hosts: human, chimpanzee, and bonobo. Zoonosis (nonhuman to human transmission) and anthroponosis (human to nonhuman transmission) may play significant roles in the emergence of human adenoviral pathogens.
... As the molecular tools and developmental strategies available for the generation of non-human adenoviruses, chimpanzee adenoviruses have emerged as attractive platforms for vaccine design [185,186]. The growing popularity of chimpanzee adenoviruses as vaccine vectors is due to their ability to bypass the negative impact of pre-existing adenovirus antibodies on immunity while inducing immune responses similar to or more potent than those elicited by their human adenoviral counterparts [187][188][189]. ...
Background:
Mycobacterium tuberculosis (Mtb) is considered as one of the most efficacious human pathogens. The global mortality rate of TB stands at approximately 2 million, while about 8 to 10 million active new cases are documented yearly. It is, therefore, a priority to develop vaccines that will prevent active TB. The vaccines currently used for the management of TB can only proffer a certain level of protection against meningitis, TB, and other forms of disseminated TB in children; however, their effectiveness against pulmonary TB varies and cannot provide life-long protective immunity. Based on these reasons, more efforts are channeled towards the development of new TB vaccines. During the development of TB vaccines, a major challenge has always been the lack of diversity in both the antigens contained in TB vaccines and the immune responses of the TB sufferers. Current efforts are channeled on widening both the range of antigens selection and the range of immune response elicited by the vaccines. The past two decades witnessed a significant progress in the development of TB vaccines; some of the discovered TB vaccines have recently even completed the third phase (phase III) of clinical trial.
Objective:
The objectives of this article are to discuss the recent progress in the development of new vaccines against TB; to provide an insight on the mechanism of vaccine-mediated specific immune response stimulation; and to debate the interaction between vaccines and global interventions to end TB.
... 55,173,174 First-generation SAdV vectors were based on SAdV-25 (chimpanzee, C68), SAdV-22 (Pan 5), SAdV-23 (Pan 6), and SAdV-24 (Pan 7). 175,176 Given the promising preclincial results with these vectors, characterization of hundreds of novel chimpanzee AdV ("ChAd") isolates was undertaken and promising candidates, i.e., low seroprevalence in the human population and lack of cross-neutralization with HAdVs, are being selected for further vector development. 177 Recently, three rhesus monkeyderived AdVs related to Human mastadenovirus G were isolated, vectorized, and characterized for their immunogenic properties. ...
A novel human adenovirus was isolated from a pediatric case of acute respiratory disease in Panama City, Panama in 2011. The clinical isolate was initially identified as an intertypic recombinant based on hexon and fiber gene sequencing. Based on the analysis of its complete genome sequence, the novel complex recombinant Human mastadenovirus D (HAdV-D) strain was classified into a new HAdV type: HAdV-84, and it was designated Adenovirus D human/PAN/P309886/2011/84[P43H17F84]. HAdV-D types possess usually an ocular or gastrointestinal tropism, and respiratory association is scarcely reported. The virus has a novel fiber type, most closely related to, but still clearly distant from that of HAdV-36. The predicted fiber is hypothesised to bind sialic acid with lower affinity compared to HAdV-37. Bioinformatic analysis of the complete genomic sequence of HAdV-84 revealed multiple homologous recombination events and provided deeper insight into HAdV evolution.
... In conclusion, we investigated the use of chimpanzee adenoviruses origin-AdC7-SP/E1A-ΔE3 as oncolytic virus for cancer treatment to address the disadvantages of oncolytic adenoviruses based on AdHu5. Importantly, compared to AdHu5, chimpanzee adenoviruses exhibit the lower transduction of hepatocytes [43], which could be explained by the observation that the hexon proteins derived from chimpanzee adenoviruses could not bind to blood coagulation factor X (FX) [17]. Moreover, systemic administration of AdC7-SP/E1A-ΔE3 could also inhibit tumor growth (Figure 8). ...
Among the oncolytic virotherapy, an emerging treatment for tumor, adenoviruses are widely used at present in preclinical and clinical trials. Traditionally, oncolytic adenoviruses were developed based on the human adenovirus serotype 5 (AdHu5). However, AdHu5 has the drawbacks of preexisting anti-AdHu5 immunity in most populations, and extensive sequestration of Adhu5 by the liver through hexon, blood coagulation factor X (FX), and FX receptor interactions. To tackle these problems, we explored a novel oncolytic adenovirus AdC7-SP/E1A-ΔE3 for cancer treatment. AdC7-SP/E1A-ΔE3 was constructed by replacing the E1A promoter with tumor specific promoter survivin promoter and deleting E3 region using direct cloning methods based on simian adenovirus serotype 24 (namely AdC7). We showed that AdC7-SP/E1A-ΔE3 significantly killed tumor cell lines NCI-H508 and Huh7, and inhibited tumor growth in both NCI-H508 and Huh7 xenograft tumor models. Importantly, AdC7-SP/E1A-ΔE3 exhibited the antitumor efficacy via systemic administration. Mechanistically, infected cells were killed by AdC7-SP/E1A-ΔE3 via the p53-independent mitochondrial apoptosis pathway in which phosphorylation of BAD markedly declined and the expresses of Bik significantly went up. Therefore, AdC7-SP/E1A-ΔE3 is a promising candidate for liver and colon tumor treatment.
... Over the last decade, HAdVs, and especially SAdVs, have become important tools for improving human health as gene delivery vectors for gene therapy and for vaccine development (13,14). Given the possibility of preexisting immunity against HAdVs in humans, SAdVs are a valuable alternative, as they are presumably not found in humans (15)(16)(17). ...
The genome sequence of a simian adenovirus from a cynomolgus macaque, denoted CynAdV-1, is presented here. Phylogenetic analysis supports CynAdV-1 in an independent clade, comprising a new simian adenovirus (SAdV) species. These genome data are critical for understanding the evolution and relationships of primate adenoviruses, including zoonosis and emergent human pathogens.
... With advancements in the molecular tools and developmental strategies available to generating nonhuman adenoviruses, chimpanzee adenoviruses have emerged as attractive platforms for vaccine design. 33,67 The growing popularity of chimpanzee adenoviruses as vaccine vectors is associated with their ability to bypass the negative impact of preexisting antihuman adenovirus immunity while inducing immune responses that are similar to or more potent than those elicited by their human adenoviral counterparts. 11,12,51 Currently two chimpanzee adenovirus-based TB vaccines including AdCh68Ag85A and ChAdOx1.85A are under evaluation, both designed to express an M. tuberculosis antigen Ag85A. ...
Adenoviruses represent the most widely used viral-vectored platform for vaccine design, showing a great potential in the fight against intracellular infectious diseases to which either there is a lack of effective vaccines or the traditional vaccination strategy is suboptimal. The extensive understanding of the molecular biology of adenoviruses has made the new technologies and reagents available to efficient generation of adenoviral-vectored vaccines for both preclinical and clinical evaluation. The novel adenoviral vectors including nonhuman adenoviral vectors have emerged to be the further improved vectors for vaccine design. In this review, we discuss the latest adenoviral technologies and their utilization in vaccine development. We particularly focus on the application of adenoviral-vectored vaccines in mucosal immunization strategies against mucosal pathogens including Mycobacterium tuberculosis, flu virus, and human immunodeficiency virus.
... 55,173,174 First-generation SAdV vectors were based on SAdV-25 (chimpanzee, C68), SAdV-22 (Pan 5), SAdV-23 (Pan 6), and SAdV-24 (Pan 7). 175,176 Given the promising preclincial results with these vectors, characterization of hundreds of novel chimpanzee AdV ("ChAd") isolates was undertaken and promising candidates, i.e., low seroprevalence in the human population and lack of cross-neutralization with HAdVs, are being selected for further vector development. 177 Recently, three rhesus monkeyderived AdVs related to Human mastadenovirus G were isolated, vectorized, and characterized for their immunogenic properties. ...
Recombinant vectors based on human adenovirus serotype 5 (HAdV-5) have been extensively studied in pre-clinical models and clinical trials over the last two decades. However, the thorough understanding of the HAdV-5 interaction with human subjects has uncovered major concerns about its product applicability. High vector-associated toxicity and widespread pre-existing immunity have been shown to significantly impede the effectiveness of HAdV-5 mediated gene transfer. It is therefore that the in depth knowledge attained working on HAdV-5 is currently being used to develop alternative vectors. Here, we provide a comprehensive overview of data obtained in recent years disqualifying the HAdV-5 vector for systemic gene delivery as well as novel strategies being pursued to overcome the limitations observed with particular emphasis on the ongoing vectorization efforts to obtain vectors based on alternative serotypes.Molecular Therapy (2015); doi:10.1038/mt.2015.194.
... For example, vectors have been recently developed from chimpanzee Ads C1 or C8 (AdC). Initially AdC vectors gained attention since human sera fail to significantly neutralize them (Roy et al. 2004). Notably, unlike some other serotypes, the E1-deleted mutant of AdC7 is easily propagated in vitro ). ...
Emerging and reemerging infectious diseases as well as cancer pose
great global health impacts on the society. Vaccines have emerged as effective
treatments to prevent or reduce the burdens of already developed diseases. This is
achieved by means of activating various components of the immune system to
generate systemic inflammatory reactions targeting infectious agents or diseased
cells for control/elimination. DNA virus-based genetic vaccines gained significant
attention in the past decades owing to the development of DNA manipulation
technologies, which allowed engineering of recombinant viral vectors encoding
sequences for foreign antigens or their immunogenic epitopes as well as various
immunomodulatory molecules. Despite tremendous progress in the past 50 years,
many hurdles still remain for achieving the full clinical potential of viral-vectored
vaccines. This chapter will present the evolution of vaccines from “live”
or “attenuated” first-generation agents to recombinant DNA and viral-vectored
vaccines. Particular emphasis will be given to human adenovirus (Ad) for the
development of prophylactic and therapeutic vaccines. Ad biological properties
related to vaccine development will be highlighted along with their advantages and
potential hurdles to be overcome. In particular, we will discuss (1) genetic modifications
in the Ad capsid protein to reduce the intrinsic viral immunogenicity,
(2) antigen capsid incorporation for effective presentation of foreign antigens to the
immune system, (3) modification of the hexon and fiber capsid proteins for Ad liver
de-targeting and selective retargeting to cancer cells, (4) Ad-based vaccines carrying
“arming” transgenes with immunostimulatory functions as immune adjuvants,
and (5) oncolytic Ad vectors as a new therapeutic approach against cancer. Finally,
the combination of adenoviral vectors with other non-adenoviral vector systems,
the prime/boost strategy of immunization, clinical trials involving Ad-based vaccines,
and the perspectives for the field development will be discussed.
... These nonhuman Ad vectors have been developed from multiple species, including, bovine, canine, chimpanzee and porcine [67]. Vectors derived from chimpanzee Ads C1 or C8 (AdC), have been recently developed, initially these vectors gained popularity since it was demonstrated that human sera does not significantly neutralize AdC vectors [132]. Importantly, unlike some other serotypes, the E1-deleted version of AdC7 is easily propagated [133]. ...
To expand on knowledge gained from previous antigen capsid-incorporation studies, our group set out to create novel vaccine vectors that would yield optimal vaccine efficacy by maximizing the size of antigens which could be incorporated within the capsid protein, hexon. Our 2008 manuscript evaluated the use of Ad5 HVR2 or HVR5 vectors containing identical antigenic epitopes in either region. To compare the capacities and flexibility of Ad5 HVR2 to those of HVR5, we genetically incorporated identical epitopes of increasing size within HVR2 or HVR5 of the Ad5 hexon. The epitopes ranged in size from 33-83 amino acids. Stable vectors were produced with incorporations of 33 amino acids plus a 12 amino acid linker at HVR2 or HVR5. In addition, stable vectors were produced with incorporations up to 53 amino acids plus a 12 amino acid linker in HVR5. With respect to the selected antigens, HVR5 was more permissive, allowing an epitope incorporation of 65 amino acids. Whole virus enzyme-linked immunosorbent assay (ELISA) analysis revealed that the model antigens were virion surface-exposed, and in vivo immunization with these vectors elicited antigen-specific immune responses [93].
... Gene delivery by ChAdV-68 is CAR dependent (Cohen et al., 2002), and unlike the E1 of HAdV-5, the flanking sequences of ChAdV-68 are nonhomologous with cell-derived E1, preventing the formation of replication-competent viruses . Development of vectors derived from chimpanzee AdVs Pan 5 (ChAdV-5/SAdV-22), Pan 6 (ChAdV-6/SAdV-23), and Pan 7 (ChAdV-7/SAdV-24), members of HAdV E species, started in 2004 (Roy et al., 2004). The vectors transduced skeletal muscle with the same efficiency as HAdV-5 vectors, but without being neutralized by human sera, and the neutralization domains showed to be different in Pan 6 and Pan 7 both in vitro and in vivo. ...
Adenoviruses are efficient gene delivery vectors based on their ability to transduce a wide variety of cell types and drive high-level transient transgene expression. While there have been advances in modifying human adenoviral (HAdV) vectors to increase their safety profile, there are still pitfalls that need to be further addressed. Preexisting humoral and cellular immunity against common HAdV serotypes limits the efficacy of gene transfer and duration of transgene expression. As an alternative, nonhuman AdV (NHAdV) vectors can circumvent neutralizing antibodies against HAdVs in immunized mice and monkeys and in human sera, suggesting that NHAdV vectors could circumvent preexisting humoral immunity against HAdVs in a clinical setting. Consequently, there has been an increased interest in developing NHAdV vectors for gene delivery in humans. In this review, we outline the recent advances and limitations of HAdV vectors for gene therapy and describe examples of NHAdV vectors focusing on their immunogenicity, tropism, and potential as effective gene therapy vehicles.
... Paradoxically, despite this role as a human pathogen, HAdVs are also important biomedical tools as vectors for epitope and gene delivery (Darr et al., 2009;Fujishiro et al., 2005;Graham and Prevec, 1992;Stone et al., 2006). To bypass pre-existing immune responses, recent attention to non-human simian adenoviruses (SAdVs) as potential gene vectors has serendipitously brought additional genomes into the data set (Roy et al., 2004a;Roy et al., 2004b;Roy et al., 2012;Roy et al., 2009;Roy et al., 2006). Presented in this report is the computational analysis of a pair of nearly identical SAdV genomes from a chimpanzee and a bonobo (Roy et al., 2009). ...
Emergent human and simian adenoviruses (HAdVs) may arise from genome recombination. Computational analysis of SAdV type 35 reveals a genome comprising a chassis with elements mostly from two simian adenoviruses, SAdV-B21 and -B27, and regions of high sequence similarity shared with HAdV-B21 and HAdV-B16. Although recombination direction cannot be determined, the presence of these regions suggests prior infections of humans by an ancestor of SAdV-B35, and/or vice versa. Absence of this virus in humans may reflect non-optimal conditions for zoonosis or incomplete typing, e.g., limited epitope-based. The presence of both a critical viral replication element found in HAdV genomes and genes that are highly similar to ones in HAdVs suggest the potential to establish in a human host. This allows a prediction that this virus may be a nascent human respiratory pathogen. The recombination potential of human and simian adenovirus genomes should be considered in the use of SAdVs as vectors for gene delivery in humans.
... Adenoviral vectors can efficiently transfer foreign genes into lung tissue in vivo [24,25]. However, adenovirus-mediated gene expression is transient and may be associated with a significant inflammatory response, depending on the viral dose [26]. Unfortunately, this inflammatory response limits the use of these vectors in treating acute lung injury. ...
In this study, we cloned human KGF (hKGF) genes using RT-PCR techniques and developed a eukaryotic expression plasmid vector capable of directing the expression of functional hKGF. Monolayer culture of human embryo lung fibro-blast (HLF) was used for isolation of total RNA. Then the total RNA was purified and reverse- transcribed into cDNA using an oligo (dT) primer. A full PCR fragment for hKGF was generated and cloned. Restriction digestion and nucleo-tide sequence analysis validated the complete hKGF transcription. The hKGF cDNA fragment was inserted into pEGFP-C2 vector by means of recombinant DNA technology and verified by restriction analysis and sequencing. We have constructed pEGFP-C2-hKGF encoding the green fluorescent protein (GFP). Furthermore, hKGF had the effect on AEC II proliferation. These results suggest that the potential appli-cation of a hKGF plasmid of gene expression should be useful for sustained AEC proliferation, and its in vivo efficacy needs to be validated.
Keywords:
... Serendipitously, the Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/yviro Virology recent attention to non-human simian adenoviruses (SAdVs) in this capacity has brought additional genomes into the data set (Roy et al., 2004a(Roy et al., , 2004b(Roy et al., , 2012(Roy et al., , 2009(Roy et al., , 2006, complementing the HAdV genomes and allowing for higher resolution into origins of other HAdVs. One example is HAdV-E4, which has been shown to contain a genome with relatively high similarity to several chimpanzee adenoviruses (Purkayastha et al., 2005a). ...
Computational analysis of human adenovirus type 4 (HAdV-E4), a pathogen that is the only HAdV member of species E, provides insights into its zoonotic origin and molecular adaptation. Its genome encodes a domain of the major capsid protein, hexon, from HAdV-B16 recombined into the genome chassis of a simian adenovirus. Genomes of two recent field strains provide a clue to its adaptation to the new host: recombination of a NF-I binding site motif, which is required for efficient viral replication, from another HAdV genome. This motif is absent in the chimpanzee adenoviruses and the HAdV-E4 prototype, but is conserved amongst other HAdVs. This is the first report of an interspecies recombination event for HAdVs, and the first documentation of a lateral partial gene transfer from a chimpanzee AdV. The potential for such recombination events are important when considering chimpanzee adenoviruses as candidate gene delivery vectors for human patients.
... Indeed, it has been shown that the same processes can be applied to ChAd, leading to productivities above 1 × 10 10 vp/ml (i.e., >one dose/ml). Importantly, it has been shown that replication-competent Ad are not generated when ChAd are propagated in HEK293, due to insufficient sequence homology between the ChAd E1 flanking regions and those of the cell-resident E1 [55,111]. Recombination events leading to replication-competent adenoviruses generation cannot occur in PER.C6 cells given that the cell-resident E1 sequences have been engineered not to overlap with Ad5 E1 region. ...
Replication-defective chimpanzee adenovirus vectors are emerging as a promising new class of genetic vaccine carriers. Chimpanzee adenovirus vectors have now reached the clinical stage and appear to be endowed with all the properties needed for human vaccine development, including high quality and magnitude of the immune response induced against the encoded antigens, good safety and ease of manufacturing on a large-scale basis. Here the authors review the recent findings of this novel class of adenovirus vectors and compare their properties to other clinical stage vaccine vectors derived from poxvirus, alphavirus and human adenovirus.
... Recombinant adenovirus vectors expressing NP [19] or both NP and M2 [20,21] can protect mice against a range of influenza virus challenges, including highly pathogenic avian H5N1 strains. While potential interference by prior immunity to human adenoviruses has been suggested as a barrier, this issue can be circumvented by use of vectors based on animal adenoviruses22232425. Chimpanzee adenoviruses have been shown to be useful vaccine vectors in a variety of animal studies2627282930, and the prevalence of neutralizing antibodies against chimpanzee adenoviruses is low in human populations313233, but not all of them are equally immunogenic. ...
Among approximately 1000 adenoviruses from chimpanzees and bonobos studied recently, the Pan Adenovirus type 3 (PanAd3, isolated from a bonobo, Pan paniscus) has one of the best profiles for a vaccine vector, combining potent transgene immunogenicity with minimal pre-existing immunity in the human population. In this study, we inserted into a replication defective PanAd3 a transgene expressing a fusion protein of conserved influenza antigens nucleoprotein (NP) and matrix 1 (M1). We then studied antibody and T cell responses as well as protection from challenge infection in a mouse model. A single intranasal administration of PanAd3-NPM1 vaccine induced strong antibody and T cell responses, and protected against high dose lethal influenza virus challenge. Thus PanAd3 is a promising candidate vector for vaccines, including universal influenza vaccines.
... The ME string contains the BALB/c H-2Kd epitope Pb9 (CS252–260, SYIPSAEKI) from the P. berghei malaria CS protein among a number of other human B- and T-cell epitopes.38 AdC7, AdC9, ChAd63, AdH5, and MVA vectors expressing ME.TRAP were constructed and propagated as described previously.6,12,39 Design and construction of Ad and MVA vectors expressing the M. tuberculosis Ag 85A has been described earlier.40,41,42 ...
Substantial protection can be provided against the pre-erythrocytic stages of malaria by vaccination first with an adenoviral and then with an modified vaccinia virus Ankara (MVA) poxviral vector encoding the same ME.TRAP transgene. We investigated whether the two vaccine components adenovirus (Ad) and MVA could be coinjected as a mixture to enhance protection against malaria. A single-shot mixture at specific ratios of Ad and MVA (Ad+MVA) enhanced CD8+ T cell-dependant protection of mice against challenge with Plasmodium berghei. Moreover, the degree of protection could be enhanced after homologous boosting with the same Ad+MVA mixture to levels comparable with classic heterologous Ad prime-MVA boost regimes. The mixture increased transgene-specific responses while decreasing the CD8+ T cell antivector immunity compared to each vector used alone, particularly against the MVA backbone. Mixed vector immunization led to increased early circulating interferon-γ (IFN-γ) response levels and altered transcriptional microarray profiles. Furthermore, we found that sequential immunizations with the Ad+MVA mixture led to consistent boosting of the transgene-specific CD8+ response for up to three mixture immunizations, whereas each vector used alone elicited progressively lower responses. Our findings offer the possibility of simplifying the deployment of viral vectors as a single mixture product rather than in heterologous prime-boost regimens.
... Furthermore, one of the macaques had preexisting serum antibodies that reacted with Ad35K++, likely due to immunological crossreactivity between simian and human adenoviruses. 30 In spite of this, Ad35K++ enhanced rituximab-mediated B-cell depletion. ...
We have developed a technology that depletes the complement regulatory protein (CRP) CD46 from the cell surface, and thereby sensitizes tumor cells to complement-dependent cytotoxicity triggered by therapeutic monoclonal antibodies (mAbs). This technology is based on a small recombinant protein, Ad35K++, which induces the internalization and subsequent degradation of CD46. In preliminary studies, we had demonstrated the utility of the combination of Ad35K++ and several commercially available mAbs such as rituximab, alemtuzumab, and trastuzumab in enhancing cell killing in vitro as well as in vivo in murine xenograft and syngeneic tumor models. We have completed scaled manufacturing of Ad35K++ protein in Escherichia coli for studies in nonhuman primates (NHPs). In macaques, we first defined a dose of the CD20-targeting mAb rituximab that did not deplete CD20-positive peripheral blood cells. Using this dose of rituximab, we then demonstrated that pretreatment with Ad35K++ reconstituted near complete elimination of B cells. Further studies demonstrated that the treatment was well tolerated and safe. These findings in a relevant large animal model provide the rationale for moving this therapy forward into clinical trials in patients with CD20-positive B-cell malignancies.Molecular Therapy (2012); doi:10.1038/mt.2012.212.
... Simian adenoviruses (SAdV) isolated from great apes are not phylogenetically distinct from human adenoviruses (HAdV), and group together into the same viral species (Human adenovirus B, C and E) [27]. Although HAdV-4 is the sole representative of Human adenovirus E derived from humans, many of the chimpanzee adenoviruses group phylogenetically within species E, including vaccine vector candidates ChAd63, AdC68 (SAdV-25), AdC7 (SAdV-24) and AdC6 (SAdV-23) [5,18,28,29]. Phylogenetic trees were constructed based on the DNA sequences of hexon and fiber proteins since these are the major surface-exposed capsid components and are believed to be the primary determinants of vector tropism and serum neutralisation (Figure 1). The phylogenetic analysis indicates that chimpanzee adenovirus Y25 also groups with Human adenovirus E viruses. ...
Recombinant adenoviruses are among the most promising tools for vaccine antigen delivery. Recently, the development of new vectors has focused on serotypes to which the human population is less exposed in order to circumvent pre-existing anti vector immunity. This study describes the derivation of a new vaccine vector based on a chimpanzee adenovirus, Y25, together with a comparative assessment of its potential to elicit transgene product specific immune responses in mice. The vector was constructed in a bacterial artificial chromosome to facilitate genetic manipulation of genomic clones. In order to conduct a fair head-to-head immunological comparison of multiple adenoviral vectors, we optimised a method for accurate determination of infectious titre, since this parameter exhibits profound natural variability and can confound immunogenicity studies when doses are based on viral particle estimation. Cellular immunogenicity of recombinant E1 E3-deleted vector ChAdY25 was comparable to that of other species E derived chimpanzee adenovirus vectors including ChAd63, the first simian adenovirus vector to enter clinical trials in humans. Furthermore, the prevalence of virus neutralizing antibodies (titre >1:200) against ChAdY25 in serum samples collected from two human populations in the UK and Gambia was particularly low compared to published data for other chimpanzee adenoviruses. These findings support the continued development of new chimpanzee adenovirus vectors, including ChAdY25, for clinical use.
... Adenoviral vectors can efficiently transfer foreign genes into lung tissue in vivo [24,25]. However, adenovirus-mediated gene expression is transient and may be associated with a significant inflammatory response, depending on the viral dose [26] . Unfortunately , this inflammatory response limits the use of these vectors in treating acute lung injury. ...
In this study, we cloned human KGF (hKGF) genes using RT-PCR techniques and developed a eukaryotic expression plasmid vector capable of directing the expression of functional hKGF. Monolayer culture of human embryo lung fibro-blast (HLF) was used for isolation of total RNA. Then the total RNA was purified and reverse-transcribed into cDNA using an oligo (dT) primer. A full PCR fragment for hKGF was generated and cloned. Restriction digestion and nucleo-tide sequence analysis validated the complete hKGF transcription. The hKGF cDNA fragment was inserted into pEGFP-C2 vector by means of recombinant DNA technology and verified by restriction analysis and sequencing. We have constructed pEGFP-C2-hKGF encoding the green fluorescent protein (GFP). Furthermore, hKGF had the effect on AEC II proliferation. These results suggest that the potential appli-cation of a hKGF plasmid of gene expression should be useful for sustained AEC proliferation, and its in vivo efficacy needs to be validated.
... The ME string contains the BALB/c H-2K d epitope Pb9, among a number of other human B and T cell epitopes (23). AdC9 was constructed and propagated as described previously (24). ...
Identification of correlates of protection for infectious diseases including malaria is a major challenge and has become one of the main obstacles in developing effective vaccines. We investigated protection against liver-stage malaria conferred by vaccination with adenoviral (Ad) and modified vaccinia Ankara (MVA) vectors expressing pre-erythrocytic malaria Ags. By classifying CD8(+) T cells into effector, effector memory (T(EM)), and central memory subsets using CD62L and CD127 markers, we found striking differences in T cell memory generation. Although MVA induced accelerated central memory T cell generation, which could be efficiently boosted by subsequent Ad administration, it failed to protect against malaria. In contrast, Ad vectors, which permit persistent Ag delivery, elicit a prolonged effector T cell and T(EM) response that requires long intervals for an efficient boost. A preferential T(EM) phenotype was maintained in liver, blood, and spleen after Ad/MVA prime-boost regimens, and animals were protected against malaria sporozoite challenge. Blood CD8(+) T(EM) cells correlated with protection against malaria liver-stage infection, assessed by estimation of number of parasites emerging from the liver into the blood. The protective ability of Ag-specific T(EM) cells was confirmed by transfer experiments into naive recipient mice. Thus, we identify persistent CD8 T(EM) populations as essential for vaccine-induced pre-erythrocytic protection against malaria, a finding that has important implications for vaccine design.
... However, some serotypes of Ad are not dependent on CAR to target the cell, such as Adhu35 and chimpanzee Ad C1, which use CD46 for initial binding [20]. CAR independent Ad can be generated as a replicating viral vector in cancer gene therapy with efficient target transduction and efficient replication, as shown in Fig. 2. Chimpanzee Ad has a comparable excellent immunogenicity to AdHu5 virus, and also has many serotypes that could be used for heterologous re-administration to bypass pre-existing neutralizing antibodies in vivo [21,22]. Our lab has developed Ad vectors including rare human serotype such as AdHu26 and Ads from chimpanzee [23,24]. ...
Viral delivery for cancer gene therapy is a promising approach, where traditional radiotherapy or chemotherapy to limit proliferation and movement of cancer cells has met resistance. Based on the new understanding of the biology of the viral vectors, therapeutic viral vectors for cancer gene therapy have been improved for greater safety and efficacy as well as transitioned from being non-replicating to replication-competent. Traditional oncolytic vectors have focused on eliminating tumor growth, while novel vectors simultaneously target epithelial-to-mesenchymal transition (EMT) in cancer cells, which could further prevent and reverse the aggressive tumor progression. In this review, we highlight the illustrative examples of cancer gene therapy in clinical trials as well as preclinical data and include proposals on methods to further enhance the safety and efficacy of oncolytic viral vectors in cancer gene therapy.
... We have investigated the use of adenovirus gene transfer vectors based on adenoviruses derived from non-human sources because the prevalence of antibodies capable of neutralizing these viruses is probably low in humans (Roy et al., 2004(Roy et al., , 2006. We screened for simian adenoviruses where the E1 deletions could be complemented in cell lines such as HEK 293 that are currently used to manufacture adenovirus vectors, i.e. their growth characteristics would not restrict them to grow in simian-derived cell lines. ...
The complete nucleotide sequence of an isolate of simian adenovirus 7 (SAdV-7) was determined. The genome organization of this isolate was found to be similar to that of other primate adenoviruses with two principal notable points: severe truncation of the E1A and E1B 19K proteins and an E3 region encoding only the 12.5K homologue. The viral gene products of SAdV-7 are most closely related to simian adenovirus 1 (SAdV-1), and like SAdV-1, are related to the human adenovirus species HAdV-F, such as the enteric adenoviruses HAdV-40 and HAdV-41 and the recently defined HAdV-G (HAdV-52). Two kinds of gene transfer vectors were made: a replication-competent SAdV-7-based vector with no genomic deletion, and a standard replication-incompetent vector deleted for E1. Importantly, the E1-deleted vector could be propagated to high titre by trans-complementation in human HEK 293 cells.
... Purified E1-deleted Ad vectors expressing gag of HIV-1 clade B, derived from simian serotypes C6 (AdC6) or C68 (AdC68) were produced and quality controlled as described [8, 35]. ...
Most studies on E1-deleted adenovirus (Ad) vectors as vaccine carriers for antigens of HIV-1 have focused on induction of central immune responses, although stimulation of mucosal immunity at the genital tract (GT), the primary port of entry of HIV-1, would also be highly desirable. In this study, different immunization protocols using chimpanzee-derived adenoviral (AdC) vectors expressing Gag of HIV-1 clade B given in heterologous prime-boost regimens were tested for induction of systemic and genital immune responses. Although i.n. immunization stimulated CD8(+) T-cell responses that could be detected in the GT, this route induced only marginal cellular responses in systemic tissues and furthermore numbers of Gag-specific CD8(+) T cells contracted sharply within a few weeks. On the contrary, i.m. immunization induced higher and more sustained frequencies of vaccine-induced cells which could be detected in the GT as well as systemic compartments. Antigen-specific CD8(+) T cells could be detected 1 year after immunization in all compartments analyzed. Genital memory cells secreted IFN-γ, expressed high levels of CD103 and their phenotypes were consistent with a state of activation. Taken together, the results presented here show that i.m. vaccination with chimpanzee-derived (simian) adenovirus vectors is a suitable strategy to induce a long-lived genital CD8(+) T-cell response.
... Preexisting neutralizing antibodies dampen gene transfer efficacy and increase vector-mediated toxicity 2 . Vectors based on rare human Ad serotypes and Ad from other species are being explored to overcome the impact of pre-existing immunity [1][2][3][4][5][6] . ...
Adenoviral vectors have shown great promise as vaccine carriers and in gene transfer to correct underlying genetic diseases. Traditionally, construction of adenoviral vectors is complex and time consuming. In this paper, we provide an improved method for efficient generation of novel adenoviral vectors by using direct cloning. We introduce a feasible and detailed protocol for the development of chimpanzee adenoviruses (Ads) as molecular clones, as well as for the generation of recombinant virus from the molecular clones. Recombinant viruses are genetically stable and induce potent immune responses in animals. Generation of new Ad molecular clones or new recombinant Ad can be achieved in 2 months or 2 weeks, respectively.
... Construction of the molecular clone of AdHu26 and the recombinant vectors is described in the Results section. Construction of other molecular clones and vectors has been described previously (16, 21, 23, 25, 28). Ad vectors from which E1 was deleted expressing green fluorescent protein (GFP), Gag of HIV-1, or the rabies virus glycoprotein (rab.gp) ...
In order to better understand the broad applicability of adenovirus (Ad) as a vector for human vaccine studies, we compared four adenovirus (Ad) vectors from families C (Ad human serotype 5 [HAdV-5; here referred to as AdHu5]), D (HAdV-26; here referred to as AdHu26), and E (simian serotypes SAdV-23 and SAdV-24; here referred to as chimpanzee serotypes 6 and 7 [AdC6 and AdC7, respectively]) of the Adenoviridae. Seroprevalence rates and titers of neutralizing antibodies to the two human-origin Ads were found to be higher than those reported previously, especially in countries of sub-Saharan Africa. Conversely, prevalence rates and titers to AdC6 and AdC7 were markedly lower. Healthy human adults from the United States had readily detectable circulating T cells recognizing Ad viruses, the levels of which in some individuals were unexpectedly high in response to AdHu26. The magnitude of T-cell responses to AdHu5 correlated with those to AdHu26, suggesting T-cell recognition of conserved epitopes. In mice, all of the different Ad vectors induced CD8(+) T-cell responses that were comparable in their magnitudes and cytokine production profiles. Prime-boost regimens comparing different combinations of Ad vectors failed to indicate that the sequential use of Ad vectors from distinct families resulted in higher immune responses than the use of serologically distinct Ad vectors from the same family. Moreover, the transgene product-specific antibody responses induced by the AdHu26 and AdC vectors were markedly lower than those induced by the AdHu5 vector. AdHu26 vectors and, to a lesser extent, AdC vectors induced more potent Ad-neutralizing antibody responses. These results suggest that the potential of AdHu26 as a vaccine vector may suffer from limitations similar to those found for vectors based on other prevalent human Ads.
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.
Limonin is a natural tetracyclic triterpenoid compound, which widely exists in Euodia rutaecarpa (Juss.) Benth., Phellodendron chinense Schneid., and Coptis chinensis Franch. Its extensive pharmacological effects have attracted considerable attention in recent years. However, there is no systematic review focusing on the pharmacology, toxicity, and pharmacokinetics of limonin. Therefore, this review aimed to provide the latest information on the pharmacology, toxicity, and pharmacokinetics of limonin, exploring the therapeutic potential of this compound and looking for ways to improve efficacy and bioavailability. Limonin has a wide spectrum of pharmacological effects, including anti-cancer, anti-inflammatory and analgesic, anti-bacterial and anti-virus, anti-oxidation, liver protection properties. However, limonin has also been shown to lead to hepatotoxicity, renal toxicity, and genetic damage. Moreover, limonin also has complex impacts on hepatic metabolic enzyme. Pharmacokinetic studies have demonstrated that limonin has poor bioavailability, and the reduction, hydrolysis, and methylation are the main metabolic pathways of limonin. We also found that the position and group of the substituents of limonin are key in affecting pharmacological activity and bioavailability. However, some issues still exist, such as the mechanism of antioxidant activity of limonin not being clear. In addition, there are few studies on the toxicity mechanism of limonin, and the effects of limonin concentration on pharmacological effects and toxicity are not clear, and no researchers have reported any ways in which to reduce the toxicity of limonin. Therefore, future research directions include the mechanism of antioxidant activity of limonin, how the concentration of limonin affects pharmacological effects and toxicity, finding ways to reduce the toxicity of limonin, and structural modification of limonin—one of the key methods necessary to enhance pharmacological activity and bioavailability.
Introduction: In recent years, replication-defective chimpanzee-derived adenoviruses have been extensively evaluated as genetic vaccines. These vectors share desirable properties with human adenoviruses like the broad tissue tropism and the ease of large-scale manufacturing. Additionally, chimpanzee adenoviruses have the advantage to overcome the negative impact of pre-existing anti-human adenovirus immunity.
Areas covered: Here the authors review current pre-clinical research and clinical trials that utilize chimpanzee-derived adenoviral vectors as vaccines. A wealth of studies are ongoing to evaluate different vector backbones and administration routes with the aim of improving immune responses. The challenges associated with the identification of an optimal chimpanzee vector and immunization strategies for different immunological outcomes will be discussed.
Expert commentary: The demonstration that chimpanzee adenoviruses can be safely used in humans has paved the way to the use of a whole new array of vectors of different serotypes. However, so far no predictive signature of vector immunity in humans has been identified. The high magnitude of T cell responses elicited by chimpanzee adenoviruses has allowed dissecting the qualitative aspects that may be important for protective immunity. Ultimately, only the results from the most clinically advanced products will help establish the efficacy of the vaccine vector platform in the field of disease prevention.
Replication incompetent human adenovirus serotype 5 (HAdV-C5) has been extensively used as a delivery vehicle for gene therapy proteins and infectious disease antigens. These vectors infect replicating and nonreplicating cells, have a broad tissue tropism, elicit high immune responses and are easily purified to high titers. However, the utility of HAdV-C5 vectors as potential vaccines is limited due to pre-existing immunity within the human population that significantly reduces the immunogenicity of HAdV-C5 vaccines. In recent years, adenovirus vaccine development has focused on simian-derived adenoviral vectors, which have the desirable vector characteristics of HAdV-C5 but with negligible seroprevalence in the human population. Here, we discuss recent advances in simian adenovirus vaccine vector development and evaluate current research specifically focusing on clinical trial data.
Infection with adenovirus or exposure to adenovirus vectors induces antibodies that can interfere with gene therapy. Unfortunately, the most popular and widely studied adenovirus (human type 5) also has one of the highest prevalences of preexisting antibodies in humans. This chapter will review the surprisingly diverse mechanisms by which antibodies inhibit adenoviruses, the impact of antibodies on adenovirus vectors in vivo, and the many creative strategies that have been developed to try to mitigate the impact of antibodies on adenovirus vectors.
Viral vectors have been developed as vaccine platforms for a number of pathogens and tumors. In particular, adenovirus (Ad)-based vectors expressing genes coding for pathogen or tumor antigens have proven efficacious to induce protective immunity. Major challenges in the use of Ad vectors are the high prevalence of anti-Ad immunity and the recent observation during an Ad-based HIV vaccine trial that led to increased HIV-1 acquisition in the presence of circulating anti-Ad5 neutralizing antibodies. In this review we summarize strategies to address these challenges and focus on modifications of the Ad capsid to enhance the adjuvant effect of anti-Ad immunogenicity and to circumvent pre-existing immunity. In addition, we summarize the current status and potential of other viral vector vaccines based on adeno-associated viruses, lentiviruses and poxviruses.
Damaged cartilage has a limited power of regeneration. Although present surgical approaches are often effective, they can’t
restore articular cartilage to its normal structure. Cartilage regeneration can be stimulated by gene transfer using vectors.
Early studies show how gene therapy is a promising but feasible tool to repair cartilage damage.
Since its discovery and characterization in the early 1980s as a virus that attacks the immune system, there has been some success for the treatment of human immunodeficiency virus-1 (HIV-1) infection. However, due to the overwhelming public health impact of this virus, a vaccine is needed urgently. Despite the tireless efforts of scientist and clinicians, there is still no safe and effective vaccine that provides sterilizing immunity. A vaccine that provides sterilizing immunity against HIV infection remains elusive in part due to the following reasons: 1) degree of diversity of the virus, 2) ability of the virus to evade the hosts' immunity, and 3) lack of appropriate animal models in which to test vaccine candidates. There have been several attempts to stimulate the immune system to provide protection against HIV-infection. Here, we will discuss attempts that have been made to induce sterilizing immunity, including traditional vaccination attempts, induction of broadly neutralizing antibody production, DNA vaccines, and use of viral vectors. Some of these attempts show promise pending continued research efforts.
We recently reported the isolation and sequencing of 30 novel adenoviruses from chimpanzees, bonobos and gorillas. These adenoviruses are promising candidates for the purpose of expanding the repertoire of adenoviral serotypes that can be used to create vectors for circumventing pre-existing neutralizing antibodies in human populations. We thus aimed to create vectors from 20 of the newly isolated adenoviruses.
Plasmid molecular clones were created that harbored the complete E1-deleted genomes from 20 of the newly isolated ape adenoviruses belonging to species B, C and E. The plasmids were transfected into human embryonic kidney (HEK) 293 cells to rescue vectors. We also tested normal human sera to determine the extent of pre-existing cross-neutralizing anti-adenovirus neutralizing antibodies.
Twelve vectors could be rescued and expanded following transfection into HEK 293 cells with yields (from fifty 150-mm culture dishes) that ranged from 3 × 10(11) to 7 × 10(13) viral particles. Sera from 50 normal human donors were tested for the presence of neutralizing activity against 21 of the newly isolated ape adenoviruses. Cross-neutralizing activity was generally low, although outliers with high neutralizing activity were frequently detected. Species B ape adenoviruses generally showed the least cross-neutralization with antibodies present in the human sera that were tested.
E1-deleted adenovirus vectors can be created from a wide variety of ape adenoviruses that can be rescued and propagated in HEK 293 cells. The prevalence of pre-existing antibodies that can neutralize these adenoviruses in human populations is low.
Replication-deficient adenoviral (Ad) vectors are an attractive platform for a vaccine against lung infections caused by Pseudomonas aeruginosa. Ad vectors based on non-human serotypes have been developed to circumvent the problem of pre-existing anti-Ad immunity in humans. The present study analyzes the anti-P. aeruginosa systemic and lung mucosal immunity elicited by a non-human primate-based AdC7 vector expressing the outer membrane protein F (AdC7OprF) of P. aeruginosa. Intramuscular immunization of mice with AdC7OprF induced similar levels of serum and mucosal anti-OprF IgG and increased levels of anti-OprF IgA in lung epithelial lining fluid (ELF) compared to immunization with a human serotype Ad5OprF vector (p>0.05). OprF-specific INF-γ in splenic T cells stimulated with OprF-pulsed syngeneic splenic dendritic cells (DC) was similar following immunization with AdC7OprF compared to Ad5OprF (p>0.05). In contrast, OprF-specific INF-γ responses in lung T cells stimulated with either spleen or lung DC were increased following immunization with AdC7OprF compared to Ad5OprF (p<0.05). Interestingly, direct administration of AdC7OprF to the respiratory tract resulted in an increase of OprF-specific IgG in serum, OprF-specific IgG and IgA in lung ELF, and OprF-specific INF-γ in lung T-cells compared to immunization with Ad5OprF, and survival following challenge with a lethal dose of P. aeruginosa. These data demonstrate that systemic or lung mucosal immunization with an AdC7-based vaccine vector induces superior pulmonary humoral and cellular anti-transgene immunity compared to immunization with an Ad5-based vector and favors AdC7-based vectors as vaccines to induce lung mucosal immunity.
Recombination between a 360-base-pair (bp) segment of a wild-type thymidine kinase gene (tk) from each of three different strains (F, MP, and 101) of herpes simplex virus type one and a complete herpes simplex virus type 1 (strain F) tk gene containing an 8-bp insertion mutation was studied. The pairs of tk sequences resided as closely linked repeats within the genome of mouse LTK- cells. The frequency of recombination between sequences exhibiting 232 bp of uninterrupted homology and containing no mismatches other than the insertion mutation was comparable to the frequency of recombination between two sequences exhibiting four additional nucleotide mismatches distributed in such a way to preserve the 232-bp stretch of contiguous homology. In contrast, the placement of only two single-nucleotide mismatches (in addition to the insertion mutation) in such a manner to reduce the longest uninterrupted homology to 134 bp resulted in a 20-fold reduction in recombination. We conclude that the rate of intrachromosomal recombination in mammalian cells is determined by the amount of uninterrupted homology available and not by the total number of mismatches within a given interval of DNA. Furthermore, efficient recombination appears to require between 134 and 232 bp of uninterrupted homology; single-nucleotide heterologies are most likely sufficient to disrupt the minimal efficient recombination target. We also observed that if recombination was allowed to initiate within sequences exhibiting perfect homology, the event could propagate through and terminate within adjacent sequences exhibiting 19% base pair mismatch. We interpret this to mean that heterology exerts most of its impact on early rather than late steps of intrachromosomal recombination in mammalian cells.
We constructed a non-defective bovine adenovirus type 3 recombinant (BAd3-Luc) containing the firefly luciferase gene inserted in the early region 3 (E3) of the BAd3 genome. Deletion of a 696 bp XhoI-NcoI E3 segment and insertion of the luciferase gene in E3 was confirmed by Southern blot analyses. Luciferase was expressed in Madin-Darby bovine kidney cells infected with BAd3-Luc as measured by enzymic assays and Western blotting. Analyses of luciferase expression in the presence or absence of 1-beta-D-arabinofuranosylcytosine indicated that approximately 70-75% of luciferase expression was dependent on viral DNA replication, suggesting that transcription of the gene was at least partially under the control of a late promoter. Although yields of infectious virus for BAd3-Luc were approximately 10-fold lower than for wild-type virus, replication of the vector was still relatively efficient. In a Western blot experiment, anti-luciferase antibody reacted with a 62 kDa protein which is of the same molecular mass as the purified firefly luciferase polypeptide. Luciferase was also expressed in the 293 cell line infected with BAd3-Luc for at least 6 days post-infection as monitored by luciferase assays. Based on these observations we suggest that BAd-based expression vectors should have excellent potential for the development of recombinant vaccines for cattle and may also be suitable as vectors for gene transfer into human cells.
In this manuscript, an E1 and E3 deleted adenoviral recombinant expressing the rabies virus glycoprotein (G protein) under the control of the cytomegalovirus early promoter was tested for induction of a rabies virus-specific immune response in mice. The construct was found to induce neutralizing antibodies and cytolytic T cells to rabies virus. Mice vaccinated with the adenoviral construct either by the systemic route or by application into the airways were protected against a subsequent infection with a virulent strain of rabies virus. The efficacy of the replication-defective construct was far superior to that of a well-characterized vaccinia rabies glycoprotein recombinant.
Preclinical studies have shown that gene transfer following readministration of viral vectors is often inefficient due to the presence of neutralizing antibodies. Vectors derived from ubiquitous human adenoviruses may have limited clinical use because preexisting humoral and cellular immunity is found in 90% of the population. Furthermore, risks associated with the use of human adenovirus vectors, such as the need to immunosuppress or tolerize patients to a potentially debilitating virus, are avoidable if efficient nonhuman adenovirus vectors are feasible. Plasmids containing recombinant canine adenovirus (CAV) vectors from which the E1 region had been deleted were generated and transfected into a CAV E1-transcomplementing cell line. Vector stocks, with titers greater than or equal to those obtained with human adenovirus vectors, were free of detectable levels of replication-competent CAV and had a low particle-to-transduction unit ratio. CAV vectors were replication defective in all cell lines tested, transduced human-derived cells at an efficiency similar to that of a comparable human adenovirus type 5 vector, and are amenable to in vivo use. Importantly, 49 of 50 serum samples from healthy individuals did not contain detectable levels of neutralizing CAV antibodies.
An adenovirus previously isolated from a mesenteric lymph node from a chimpanzee was fully sequenced and found to be similar in overall structure to human adenoviruses. The genome of this virus, called C68, is 36,521 bp in length and is most similar to subgroup E of human adenovirus, with 90% identity in most adenovirus type 4 open reading frames that have been sequenced. Substantial differences in the hexon hypervariable regions were noted between C68 and other known adenoviruses, including adenovirus type 4. Neutralizing antibodies to C68 were highly prevalent in sera from a population of chimpanzees, while sera from humans and rhesus monkeys failed to neutralize C68. Furthermore, infection with C68 was not neutralized from sera of mice immunized with human adenovirus serotypes 2, 4, 5, 7, and 12. A replication-defective version of C68 was created by replacing the E1a and E1b genes with a minigene cassette; this vector was efficiently transcomplemented by the E1 region of human adenovirus type 5. C68 vector transduced a number of human and murine cell lines. This nonhuman adenoviral vector is sufficiently similar to human serotypes to allow growth in 293 cells and transduction of cells expressing the coxsackievirus and adenovirus receptor. As it is dissimilar in regions such as the hexon hypervariable domains, C68 vector avoids significant cross-neutralization by sera directed against human serotypes.
An E1-deletion-containing adenoviral recombinant based on the chimpanzee serotype 68 (AdC68) was developed to express the
rabies virus glycoprotein. Mice immunized with this construct (AdC68rab.gp) developed antibodies to rabies virus and remained
resistant to challenge with an otherwise lethal dose of rabies virus. In naïve mice immunized intranasally, the rabies virus-specific
antibody responses elicited by AdC68rab.gp were comparable with regard to both titers and isotype profiles to those induced
by an adenoviral recombinant based on human serotype 5 (Adhu5) expressing the same transgene product. In contrast, subcutaneous
immunization with the AdC68rab.gp vaccine resulted in markedly lower antibody responses to the rabies virus glycoprotein than
the corresponding Adhu5 vaccine. Antibodies from AdC68rab.gp-immunized mice were strongly biased towards the immunoglobulin
G2a isotype. The antibody response to the rabies virus glycoprotein presented by Adhu5rab.gp was severely compromised in animals
preexposed to the homologous adenovirus. In contrast, the rabies virus-specific antibody response to the AdC68rab.gp vaccine
was at most marginally affected by preexisting immunity to common human adenovirus serotypes, such as 2, 4, 5, 7, and 12.
This novel vaccine carrier thus offers a distinct advantage over adenoviral vaccines based on common human serotypes.
A critical issue for E1-deleted adenoviral vectors manufactured from 293 cells is the emergence of replication-competent adenovirus (RCA). These contaminants arise through homologous recombination between identical sequences framing the E1 locus displayed by 293 cells, and the vector backbones. Modified recombinogenic sequences (syngen) were thus introduced within the vector backbone, and virus viability and RCA emergence were assessed. Syngen#1 is a synthetic sequence displaying silent point mutations in the pIX and IVa2 coding regions. A side by side comparison of Ad5CMV/p53 (E1-deleted adenovirus expressing the p53 tumor suppressor gene) and AVdeltaE1#1CMV/p53 (with syngen#1 in place of wild-type sequences) demonstrated a normal productivity for the modified construct. The altered sequences did not impair p53-mediated apoptosis in a model tumor cell line. Most importantly, a statistically significant decrease in terms of RCA occurrence could also be demonstrated. Degenerescence of the recombinogenic sequences could be further accentuated by modifying noncoding pIX region (syngen #2), with no effect on virus productivity and stability. We concluded that these vector modifications constitute a feasible strategy to reduce RCA emergence during amplification in 293 cells. This approach could also be applied to decrease reincorporation of the E1 genes during amplification of deltaE1deltaE4 vectors in 293/E4-trans-complementing cells.
In animal models, E1-deleted human adenoviral recombinants of the serotype 5 (AdHu5) have shown high efficacy as vaccine carriers for different Ags including those of HIV-1. Humans are infected by common serotypes of human adenovirus such as AdHu5 early in life and a significant percentage has high levels of neutralizing Abs to these serotypes, which will very likely impair the efficacy of recombinant vaccines based on the homologous virus. To circumvent this problem, a novel replication-defective adenoviral vaccine carrier based on an E1-deleted recombinant of the chimpanzee adenovirus 68 (AdC68) was developed. An AdC68 construct expressing a codon-optimized, truncated form of gag of HIV-1 induces CD8(+) T cells to gag in mice which at the height of the immune response encompass nearly 20% of the entire splenic CD8(+) T cell population. The vaccine-induced immune response provides protection to challenge with a vaccinia gag recombinant virus. Induction of transgene-specific CD8(+) T cells and protection against viral challenge elicited by the AdC68 vaccines is not strongly inhibited in animals preimmune to AdHu5 virus. However, the response elicited by the AdHu5 vaccine is greatly attenuated in AdHu5 preimmune animals.
The cellular immunogenicity of formulated plasmid DNA and replication-defective human adenovirus serotype 5 (Ad5) vaccine
vectors expressing a codon-optimized human immunodeficiency virus type 1 gag gene was examined in baboons. The Ad5 vaccine was capable of inducing consistently strong, long-lived CD8+-biased T-cell responses and in vitro cytotoxic activities. The DNA vaccine-elicited immune responses were weaker than those
elicited by the Ad5 vaccine and highly variable; formulation with chemical adjuvants led to moderate increases in the levels
of Gag-specific T cells. Increasing the DNA-primed responses with booster doses of either Ad5 or modified vaccinia virus Ankara
vaccines suggests a difference in the relative levels of cytotoxic and helper responses. The implications of these results
are discussed.
Human embryonic kidney cells have been transformed by exposing cells to sheared fragments of adenovirus type 5 DNA. The transformed cells (designated 293 cells) exhibited many of the characteristics of transformation including the elaboration of a virus-specific tumour antigen. Analysis of the polypeptides synthesized in the 293 cells by labelling with 35S-methionine and SDS PAGE showed a variable pattern of synthesis, different in a number of respects from that seen in otheruman cells. On labelling the surface of cells by lactoperoxidase catalysed radio-iodination, the absence of a labelled polypeptide analogous to the 250 K (LETS) glycoprotein was noted. Hybridization of labelled cellular RNA with restriction fragments of adenovirus type 5 DNA indicated transcription of a portion of the adenovirus genome at the conventional left hand end.
Ability of replication-defective adenovirus vectors to achieve efficient gene transfer in most of the mammalian cell types makes them useful vehicles for many gene transfer applications, including their use in assessing gene function. High throughput creation of recombinant adenovirus becomes a critical path to the expanding utility of adenovirus vector technology. Here, we report a process in which recombinant adenovirus vectors are isolated as single molecular clones through a convenient direct cloning and green-white selection procedure, and directly transfected into 293 cells where virus is rescued through an enzymatic reaction mediated by an intron-encoding rare endonuclease I-Sce I. This process of enzymatic rescue of circular molecular clones was at least 10-fold more efficient than that using linearized clones for transfection. This method will facilitate a high throughput creation of vectors as required for screening gene function.
Replication-deficient human adenovirus type 5 (Ad5) can be produced to high titers in complementing cell lines, such as PER.C6,
and is widely used as a vaccine and gene therapy vector. However, preexisting immunity against Ad5 hampers consistency of
gene transfer, immunological responses, and vector-mediated toxicities. We report the identification of human Ad35 as a virus
with low global prevalence and the generation of an Ad35 vector plasmid system for easy insertion of heterologous genes. In
addition, we have identified the minimal sequence of the Ad35-E1B region (molecular weight, 55,000 [55K]), pivotal for complementation
of fully E1-lacking Ad35 vector on PER.C6 cells. After stable insertion of the 55K sequence into PER.C6 cells a cell line
was obtained (PER.C6/55K) that efficiently transcomplements both Ad5 and Ad35 vectors. We further demonstrate that transduction
with Ad35 is not hampered by preexisting Ad5 immunity and that Ad35 efficiently infects dendritic cells, smooth muscle cells,
and synoviocytes, in contrast to Ad5.
Basnight, M., Jr. (NINDS, NIH, Bethesda, Md. 20014), N. G. Rogers, C. J. Gibbs, Jr. and D. C. Gajdusek. haracterization of four new adenovirus serotypes isolated from chimpanzee tissue explants. Amer J Epidem 94: 166-171, 1971.-Four new latent adenovirus serotypes were isolated from explanted lymph nodes of chimpanzees in the late stages of experimental kuru and Creutzfeldt-Jakob disease. These viruses induced typical adenovirus cytopathology in human embryonic kidney cell cultures and fixed complement in the presence of human adenovirus antiserum. They had the biochemical and physical characteristics of adenoviruses; they propagated better in cell cultures of human than in those of simian origin. They were not neutralized by antisera of known human and simian adenovirus types and were immunologically distinct from each other. The viruses are not believed to be the agents of the experimental kuru or Creutzfeldt-Jakob disease in the chimpanzees from which they were isolated, although these animals developed homologous neutralizing antibodies during the course of their disease. Neutralizing antibodies to these new adenovirus serotypes were also found in normal chimpanzees, but were absent from sera of animal handlers who had been in close contact with the chimpanzees for 1-3 years.
Forty-seven strains of virus have been isolated from sterile long maintained tissue explants from chimpanzees suffering from experimental kuru. Many are new simian viruses, and none have been demonstrated to be a cause of the kuru syndrome.
Recombinant adenoviruses with E1 sequences deleted efficiently transfer genes into a wide variety of target cells. Antigen- and nonantigen-specific responses to the therapy lead to toxicity, loss of transgene expression, and difficulties with vector readministration. We have created new cell lines that allowed the isolation of more disabled adenovirus vectors that have both E1 and E4 deletions. Studies with murine models of liver-directed gene therapy indicated that the E1- and E4-deleted vector expresses fewer virus proteins and induces less apoptosis, leading to blunted host responses and an improved safety profile. The impact of the E4 deletion on the stability of vector expression was confounded by immune responses to the transgene product, which in this study was beta-galactosidase. When transgene responses were eliminated, the doubly deleted vector was substantially more stable in mouse liver than was the E1-deleted construct. These studies indicate that adenovirus vectors with both E1 and E4 deletions may have advantages in terms of safety and efficacy over first-generation constructs for liver-directed gene therapy.
An ovine adenovirus which may be the prototype for a new group of adenoviruses has been engineered as a gene transfer vector. One recombinant containing a 0.95-kb insertion expressed a sheep parasite antigen from the ovine adenovirus major late promoter and tripartite leader sequences. It was shown that insertions of at least 4.3 kb were tolerated at either one of two sites in the genome without the introduction of a compensating deletion. The unique structure of this viral genome was further emphasized by the discovery that four open reading frames at the right hand end show significant identity to each other but not to other sequences in the databases. Two other unrelated open reading frames were also present. RT-PCR analysis identified two transcripts in this region which were derived from a promoter which was located very close to, or within the ITR sequence. Splicing removed all but the first and last of the ORFs from these RNAs, suggesting that some sequences might be nonessential for replication in vitro. A approximately 2-kb deletion, which removed or truncated the internal reading frames was introduced into the region without affecting virus viability. The carrying capacity of OAV recombinants should therefore be at least 6.3 kb. The relative packaging capacity of OAV (114%) therefore exceeds that of Ad5 (105%), although a comparison of virus particle sizes by electron microscopy showed that OAV was smaller than Ad5. These studies improve the potential utility of OAV as a gene transfer vector.
The human-viral junctions of integrated adenovirus type 5 (Ad5) DNA in 293 cells have been cloned and sequenced. The Ad5 sequences extend from nucleotides (nt) 1 to 4344 and are located in the pregnancy-specific beta-1-glycoprotein 4 (PSG 4) gene. This maps the insertion of Ad5 DNA to human chromosome 19 (19q13.2). The Ad5 sequences are represented as a single collinear insertion of viral DNA with no rearrangements. There were 19 bp of PSG4 DNA deleted at the site of insertion and an extra 3 nt (GTC) at the left viral/cellular junction. A short patch of homology between viral DNA and PSG4 DNA (6 nt with 1 mismatch) was present at the right junction.
Retired greyhound dogs, with low or absent antibody titers to rabies virus following previous vaccinations with commercially available vaccines, were immunized either subcutaneously or intramuscularly with a replication-defective recombinant adenovirus expressing the rabies virus glycoprotein termed Adrab.gp. Immunized animals developed high titers (geometric mean titers of 2630 and 5329) of viral neutralizing antibodies (VNA) against rabies virus by 10 days after vaccination. The antibody titers were even higher (geometric mean titers of 19349 and 122086) by 21 days after vaccination. The results indicate that the recombinant adenovirus expressing rabies virus glycoprotein is capable of inducing antibody immune responses in dogs and therefore may be developed as a rabies virus vaccine for dogs.
Recombinant adeno-associated virus (AAV) holds much promise for human gene therapy. While evidence indicates that AAV mediates long-term gene transfer in several different tissues, difficulty in preparing and purifying this viral vector in large quantities remains a major obstacle for evaluating AAV vectors in clinical trials. The current method of purification, based on sedimentation through cesium chloride, is not scaleable and yields product of insufficient quality. In this article we report a new technique for purifying AAV, using a fully closed two-column chromatography system. Yields of AAV vectors purified by this method are high, potency is increased, and the purity of column-purified preparations is substantially improved. We previously reported a novel method to generate AAV based on an AAV Rep/Cap-containing cell line (B50) and an Ad-AAV hybrid virus, which is amenable to scale-up in bioreactors. By combining the new, fully scaleable purification process we report here with the B50/hybrid production method, it would be feasible to prepare AAV vectors to the scale and purity required for clinical and potential commercial applications.
Outbreaks of haemorrhagic fever caused by the Ebola virus are associated with high mortality rates that are a distinguishing feature of this human pathogen. The highest lethality is associated with the Zaire subtype, one of four strains identified to date. Its rapid progression allows little opportunity to develop natural immunity, and there is currently no effective anti-viral therapy. Therefore, vaccination offers a promising intervention to prevent infection and limit spread. Here we describe a highly effective vaccine strategy for Ebola virus infection in non-human primates. A combination of DNA immunization and boosting with adenoviral vectors that encode viral proteins generated cellular and humoral immunity in cynomolgus macaques. Challenge with a lethal dose of the highly pathogenic, wild-type, 1976 Mayinga strain of Ebola Zaire virus resulted in uniform infection in controls, who progressed to a moribund state and death in less than one week. In contrast, all vaccinated animals were asymptomatic for more than six months, with no detectable virus after the initial challenge. These findings demonstrate that it is possible to develop a preventive vaccine against Ebola virus infection in primates.
HIV-specific T cell immune responses will play an important role in any HIV vaccine paradigm. Studies in rhesus monkeys have shown that significant and persistent virus-specific T cell responses can be elicited with vaccines incorporating viral genetic sequences and that these responses are primarily mediated by CD8 T cells. Benefits such as stable CD4 levels and viral control have resulted. Two vaccine candidates developed by Merck and Co., Inc., including a non-replicating adenoviral vector, have been studied in animals and are now being studied in Phase I clinical trials in humans. Important considerations include cross-clade reactivity (effectiveness in diverse HIV-infected populations), tolerability, and durability of response. Ongoing studies are looking at responses in both uninfected and infected individuals. Optimal vaccine combinations as well as the development and testing of vaccines with multiple genetic targets are part of future plans investigating this vaccine strategy.
Containment of highly lethal Ebola virus outbreaks poses a serious public health challenge. Although an experimental vaccine has successfully protected non-human primates against disease, more than six months was required to complete the immunizations, making it impractical to limit an acute epidemic. Here, we report the development of accelerated vaccination against Ebola virus in non-human primates. The antibody response to immunization with an adenoviral (ADV) vector encoding the Ebola glycoprotein (GP) was induced more rapidly than with DNA priming and ADV boosting, but it was of lower magnitude. To determine whether this earlier immune response could nonetheless protect against disease, cynomolgus macaques were challenged with Ebola virus after vaccination with ADV-GP and nucleoprotein (NP) vectors. Protection was highly effective and correlated with the generation of Ebola-specific CD8(+) T-cell and antibody responses. Even when animals were immunized once with ADV-GP/NP and challenged 28 days later, they remained resistant to challenge with either low or high doses of virus. This accelerated vaccine provides an intervention that may help to limit the epidemic spread of Ebola, and is applicable to other viruses.
Wilson 204 Wistar, 3601 Spruce Street Philadelphia, PA 19104 E-mail: wilsonjm@mail.med.upenn.edu Received for publication
- Roy Et
- Al
Address reprint requests to: Dr. James M. Wilson 204 Wistar, 3601 Spruce Street Philadelphia, PA 19104 E-mail: wilsonjm@mail.med.upenn.edu Received for publication January 28, 2004; accepted after re-vision March 29, 2004. Published online: April 14, 2004. ROY ET AL.
Development of a preventive vaccine for Ebola virus infection in primates
- N J Sullivan
- A Sanchez
- P E Rollin
- Z Y Yang
SULLIVAN, N.J., SANCHEZ, A., ROLLIN, P.E., YANG, Z.Y., and
NABEL, G.J. (2000). Development of a preventive vaccine for Ebola
virus infection in primates. Nature 408, 605-609.