Novel replication-incompetent vector derived from adenovirus type 11 (Ad11) for vaccination and gene therapy: low seroprevalence and non-cross-reactivity with Ad5

A novel plasmid-based adenovirus vector system that enables manufacturing of replication-incompetent (DeltaE1) adenovirus type 11 (Ad11)-based vectors is described. Ad11 vectors are produced on PER.C6/55K cells yielding high-titer vector batches after purification. Ad11 seroprevalence proves to be significantly lower than that of Ad5, and neutralizing antibody titers against Ad11 are low. Ad11 seroprevalence among human immunodeficiency virus-positive (HIV(+)) individuals is as low as that among HIV(-) individuals, independent of the level of immune suppression. The low level of coinciding seroprevalence between Ad11 and Ad35 in addition to a lack of correlation between high neutralizing antibody titers towards either adenovirus strongly suggest that the limited humoral cross-reactive immunity between these two highly related B viruses appears not to preclude the use of both vectors in the same individual. Ad11 transduces primary cells including smooth muscle cells, synoviocytes, and dendritic cells and cardiovascular tissues with higher efficiency than Ad5. Ad11 and Ad35 appear to have a similar tropism as judged by green fluorescent protein expression levels determined by using a panel of cancer cell lines. In addition, Ad5 preimmunization did not significantly affect Ad11-mediated transduction in C57BL/6 mice. We therefore conclude that the Ad11-based vector represents a novel and useful candidate gene transfer vehicle for vaccination and gene therapy.     

Recombinant adenovirus serotype 26 (Ad26) and Ad35 vaccine vectors bypass immunity to Ad5 and protect nonhuman primates against ebolavirus challenge

The use of adenoviruses (Ad) as vaccine vectors against a variety of pathogens has demonstrated their capacity to elicit strong antibody and cell-mediated immune responses. Adenovirus serotype C vectors, such as Ad serotype 5 (Ad5), expressing Ebolavirus (EBOV) glycoprotein (GP), protect completely after a single inoculation at a dose of 10(10) viral particles. However, the clinical application of a vaccine based on Ad5 vectors may be hampered, since impairment of Ad5 vaccine efficacy has been demonstrated for humans and nonhuman primates with high levels of preexisting immunity to the vector. Ad26 and Ad35 segregate genetically from Ad5 and exhibit lower seroprevalence in humans, making them attractive vaccine vector alternatives. In the series of studies presented, we show that Ad26 and Ad35 vectors generate robust antigen-specific cell-mediated and humoral immune responses against EBOV GP and that Ad5 immune status does not affect the generation of GP-specific immune responses by these vaccines. We demonstrate partial protection against EBOV by a single-shot Ad26 vaccine and complete protection when this vaccine is boosted by Ad35 1 month later. Increases in efficacy are paralleled by substantial increases in T- and B-cell responses to EBOV GP. These results suggest that Ad26 and Ad35 vectors warrant further development as candidate vaccines for EBOV.     

Alternative Serotype Adenovirus Vaccine Vectors Elicit Memory T Cells with Enhanced Anamnestic Capacity Compared to Ad5 Vectors

The failure of the adenovirus serotype 5 (Ad5) vector-based human immunodeficiency virus type 1 (HIV-1) vaccine in the STEP study has led to the development of adenovirus vectors derived from alternative serotypes, such as Ad26, Ad35, and Ad48. We have recently demonstrated that vaccines using alternative-serotype Ad vectors confer partial protection against stringent simian immunodeficiency virus (SIV) challenges in rhesus monkeys. However, phenotypic differences between the T cell responses elicited by Ad5 and those of alternative-serotype Ad vectors remain unexplored. Here, we report the magnitude, phenotype, functionality, and recall capacity of memory T cell responses elicited in mice by Ad5, Ad26, Ad35, and Ad48 vectors expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP). Our data demonstrate that memory T cells elicited by Ad5 vectors were high in magnitude but exhibited functional exhaustion and decreased anamnestic potential following secondary antigen challenge compared to Ad26, Ad35, and Ad48 vectors. These data suggest that vaccination with alternative-serotype Ad vectors offers substantial immunological advantages over Ad5 vectors, in addition to circumventing high baseline Ad5-specific neutralizing antibody titers.     

Immunogenicity of heterologous prime-boost regimens involving recombinant adenovirus serotype 11 (Ad11) and Ad35 vaccine vectors in the presence of anti-ad5 immunity

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations will likely limit the immunogenicity and clinical utility of recombinant Ad5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 and other pathogens. A potential solution to this problem is to utilize rAd vaccine vectors derived from rare Ad serotypes such as Ad35 and Ad11. We have previously reported that rAd35 vectors were immunogenic in the presence of anti-Ad5 immunity, but the immunogenicity of heterologous rAd prime-boost regimens and the extent that cross-reactive anti-vector immunity may limit this approach have not been fully explored. Here we assess the immunogenicity of heterologous vaccine regimens involving rAd5, rAd35, and novel rAd11 vectors expressing simian immunodeficiency virus Gag in mice both with and without anti-Ad5 immunity. Heterologous rAd prime-boost regimens proved significantly more immunogenic than homologous regimens, as expected. Importantly, all regimens that included rAd5 were markedly suppressed by anti-Ad5 immunity. In contrast, rAd35-rAd11 and rAd11-rAd35 regimens elicited high-frequency immune responses both in the presence and in the absence of anti-Ad5 immunity, although we also detected clear cross-reactive Ad35/Ad11-specific humoral and cellular immune responses. Nevertheless, these data suggest the potential utility of heterologous rAd prime-boost vaccine regimens using vectors derived from rare human Ad serotypes.     

A novel adenovirus type 6 (Ad6)-based hepatitis C virus vector that overcomes preexisting anti-ad5 immunity and induces potent and broad cellular immune responses in rhesus macaques

Success in resolving hepatitis C virus (HCV) infection has been correlated to vigorous, multispecific, and sustained CD8(+) T-cell response in humans and chimpanzees. The efficacy of inducing T-cell-mediated immunity by recombinant serotype 5 adenovirus vector has been proven in many animal models of infectious diseases, but its immunogenicity can be negatively influenced by preexisting immunity against the vector itself. To evaluate the less prevalent adenovirus serotype 6 (Ad6) as an alternative vector for and HCV vaccine development, we have generated serotype 5 and 6 adenoviral vectors directing expression of the nonstructural region of HCV (MRKAd5-NSmut and MRKAd6-NSmut). Immunogenicity studies in mice showed that the two vectors induced comparable T-cell responses but that only MRKAd6-NSmut was not suppressed in the presence of anti-Ad5 immunity. In contrast, preexisting anti-Ad5 immunity dramatically blunted the immunogenicity of the serotype 5-based HCV vector. Furthermore, MRKAd6-NSmut showed equivalent potency, breadth, and longevity of HCV-specific T-cell responses in rhesus macaques as the corresponding Ad5-based vector over a wide range of doses and was capable of boosting DNA-primed animals even if administered at low doses. These data support the use of the MRKAd6-NSmut for anti-HCV immunotherapy and, more generally, for the Ad6 serotype as a better genetic vaccine vehicle than Ad5.     

Adenovirus serotype 5-specific neutralizing antibodies target multiple hexon hypervariable regions

The immunogenicity of adenovirus serotype 5 (Ad5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against the hexon hypervariable regions (HVRs). We previously reported that replacing all seven HVRs with those from the rare serotype virus Ad48 resulted in a chimeric Ad5HVR48(1-7) vector that largely evaded preexisting Ad5 immunity in mice and rhesus monkeys. In this study, we evaluated the extent to which Ad5-specific NAbs are directed against various HVRs. We constructed partial HVR-chimeric Ad5 vectors with only a subset of HVRs exchanged, and we utilized these vectors in both NAb assays and murine immunogenicity studies with and without baseline Ad5 immunity. Our results demonstrate that Ad5-specific NAbs target multiple HVRs, suggesting that replacing all HVRs is required to optimize evasion of anti-Ad5 immunity. These data have important implications for the development of novel vectors for both vaccines and gene therapy.     

Development and assessment of human adenovirus type 11 as a gene transfer vector

Adenovirus vectors based on human serotype 5 (Ad5) have successfully been used as gene transfer vectors in many gene therapy-based approaches to treat disease. Despite their widespread application, many potential therapeutic applications are limited by the widespread prevalence of vector-neutralizing antibodies within the human population and the inability of Ad5-based vectors to transduce important therapeutic target cell types. In an attempt to circumvent these problems, we have developed Ad vectors based on human Ad serotype 11 (Ad11), since the prevalence of neutralizing antibodies to Ad11 in humans is low. E1-deleted Ad11 vector genomes were generated by homologous recombination in 293 cells expressing the Ad11-E1B55K protein or by recombination in Escherichia coli. E1-deleted Ad11 genomes did not display transforming activity in rodent cells. Transduction of primary human CD34+ hematopoietic progenitor cells and immature dendritic cells was more efficient with Ad11 vectors than with Ad5 vectors. Thirty minutes after intravenous injection into mice that express one of the Ad11 receptors (CD46), we found, in a pattern and at a level comparable to what is found in humans, Ad11 vector genomes in all analyzed organs, with the highest amounts in liver, lung, kidney, and spleen. Neither Ad11 genomes nor Ad11 vector-mediated transgene expression were, however, detected at 72 h postinfusion. A large number of Ad11 particles were also found to be associated with circulating blood cells. We also discovered differences in in vitro transduction efficiencies and in vivo biodistributions between Ad11 vectors and chimeric Ad5 vectors possessing Ad11 fibers, indicating that Ad11 capsid proteins other than fibers influence viral infectivity and tropism. Overall, our study provides a basis for the application of Ad11 vectors for in vitro and in vivo gene transfer and for gaining an understanding of the factors that determine Ad tropism.     

Adenovirus serotype 5 neutralizing antibodies target both hexon and fiber following vaccination and natural infection

The immunogenicity of adenovirus serotype 5 (Ad5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against the hexon hypervariable regions (HVRs). However, the role of NAbs directed against other capsid components, particularly the adenovirus fiber, remains unclear. Here we show that Ad5 NAbs target both hexon and fiber following vaccination and natural infection. Utilizing neutralization assays with capsid chimeric vectors, we observed that NAb responses to hexon appeared dominant and NAb responses against fiber were subdominant in sera from vaccinated mice, vaccinated humans, and naturally exposed humans. A novel chimeric Ad5 vector in which both the hexon HVRs and the fiber knob were exchanged nearly completely evaded Ad5-specific NAbs both in vitro and in vivo.     

Effect of neutralizing sera on factor x-mediated adenovirus serotype 5 gene transfer

The deployment of adenovirus serotype 5 (Ad5)-based vectors is hampered by preexisting immunity. When such vectors are delivered intravenously, hepatocyte transduction is mediated by the hexon-coagulation factor X (FX) interaction. Here, we demonstrate that human sera efficiently block FX-mediated cellular binding and transduction of Ad5-based vectors in vitro. Neutralizing activity correlated well with the ability to inhibit Ad5-mediated liver transduction, suggesting that prescreening patient sera in this manner accurately predicts the efficacy of Ad5-based gene therapies. Neutralization in vitro can be partially bypassed by pseudotyping with Ad45 fiber protein, indicating that a proportion of neutralizing antibodies are directed against the Ad5 fiber.     

Variability of Human Systemic Humoral Immune Responses to Adenovirus Gene Transfer Vectors Administered to Different Organs

Administration of adenovirus (Ad) vectors to immunologically naive experimental animals almost invariably results in the induction of systemic anti-Ad neutralizing antibodies. To determine if the human systemic humoral host responses to Ad vectors follow a similar pattern, we evaluated the systemic (serum) anti-Ad serotype 5 (Ad5) neutralizing antibodies in humans after administration of first generation (E1(-) E3(-)) Ad5-based gene transfer vectors to different hosts. AdGVCFTR.10 (carrying the normal human cystic fibrosis [CF] transmembrane regulator cDNA) was sprayed (8 x 10(7) to 2 x 10(10) particle units [PU]) repetitively (every 3 months or every 2 weeks) to the airway epithelium of 15 individuals with CF. AdGVCD.10 (carrying the Escherichia coli cytosine deaminase gene) was administered (8 x 10(8) to 8 x 10(9) PU; once a week, twice) directly to liver metastasis of five individuals with colon cancer and by the intradermal route (8 x 10(7) to 8 x 10(9) PU, single administration) to six healthy individuals. AdGVVEGF121.10 (carrying the human vascular endothelial growth factor 121 cDNA) was administered (4 x 10(8) to 4 x 10(9.5) PU, single administration) directly to the myocardium of 11 individuals with ischemic heart disease. Ad vector administration to the airways of individuals with CF evoked no or minimal serum neutralizing antibodies, even with repetitive administration. In contrast, intratumor administration of an Ad vector to individuals with metastatic colon cancer resulted in a robust antibody response, with anti-Ad neutralizing antibody titers of 10(2) to >10(4). Healthy individuals responded to single intradermal Ad vector variably, from induction of no neutralizing anti-Ad antibodies to titers of 5 x 10(3). Likewise, individuals with ischemic heart disease had a variable response to single intramyocardial vector administration, ranging from minimal neutralizing antibody levels to titers of 10(4). Evaluation of the data from all trials showed no correlation between the peak serum neutralizing anti-Ad response and the dose of Ad vector administered (P > 0.1, all comparisons). In contrast, there was a striking correlation between the peak anti-Ad5 neutralizing antibody levels evoked by vector administration and the level of preexisting anti-Ad5 antibodies (P = 0.0001). Thus, unlike the case for experimental animals, administration of Ad vectors to humans does not invariably evoke a systemic anti-Ad neutralizing antibody response. In humans, the extent of the response is dictated by preexisting antibody titers and modified by route of administration but is not dose dependent. Since the extent of anti-Ad neutralizing antibodies will likely modify the efficacy of administration of Ad vectors, these observations are of fundamental importance in designing human gene therapy trials and in interpreting the efficacy of Ad vector-mediated gene transfer.     

Protection against Enterovirus 71 with Neutralizing Epitope Incorporation within Adenovirus Type 3 Hexon

Enterovirus 71 (EV71) is responsible for hand, foot and mouth disease with high mortality among children. Various neutralizing B cell epitopes of EV71 have been identified as potential vaccine candidates. Capsid-incorporation of antigens into adenovirus (Ad) has been developed for a novel vaccine approach. We constructed Ad3-based EV71 vaccine vectors by incorporating a neutralizing epitope SP70 containing 15 amino acids derived from capsid protein VP1 of EV71 within the different surface-exposed domains of the capsid protein hexon of Ad3EGFP, a recombinant adenovirus type 3 (Ad3) expressing enhanced green fluorescence protein. Thermostability and growth kinetic assays suggested that the SP70 epitope incorporation into hypervariable region (HVR1, HVR2, or HVR7) of the hexon did not affect Ad fitness. The SP70 epitopes were thought to be exposed on all hexon-modified intact virion surfaces. Repeated administration of BALB/c mice with the modified Ads resulted in boosting of the anti-SP70 humoral immune response. Importantly, the modified Ads immunization of mother mice conferred protection in vivo to neonatal mice against the lethal EV71 challenge, and the modified Ads-immunized mice serum also conferred passive protection against the lethal challenge in newborn mice. Compared with the recombinant GST-fused SP70 protein immunization, immunization with the Ads containing SP70 in HVR1 or HVR2 elicited higher SP70-specific IgG titers, higher neutralization titers, and conferred more effective protection to neonatal mice. Thus, this study provides valuable information for hexon-modified Ad3 vector development as a promising EV71 vaccine candidate and as an epitope-delivering vehicle for other pathogens.     

Neutralizing antibodies and CD8+ T lymphocytes both contribute to immunity to adenovirus serotype 5 vaccine vectors

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations will likely limit the immunogenicity and clinical utility of recombinant Ad5 vector-based vaccines for human immunodeficiency virus type 1 and other pathogens. Ad5-specific neutralizing antibodies (NAbs) are thought to contribute substantially to anti-Ad5 immunity, but the potential importance of Ad5-specific T lymphocytes in this setting has not been fully characterized. Here we assess the relative contributions of Ad5-specific humoral and cellular immune responses in blunting the immunogenicity of a rAd5-Env vaccine in mice. Adoptive transfer of Ad5-specific NAbs resulted in a dramatic abrogation of Env-specific immune responses following immunization with rAd5-Env. Interestingly, adoptive transfer of Ad5-specific CD8(+) T lymphocytes also resulted in a significant and durable suppression of rAd5-Env immunogenicity. These data demonstrate that NAbs and CD8(+) T lymphocytes both contribute to immunity to Ad5. Novel adenovirus vectors that are currently being developed to circumvent the problem of preexisting anti-Ad5 immunity should therefore be designed to evade both humoral and cellular Ad5-specific immune responses.     

A Plasmodium Promiscuous T Cell Epitope Delivered within the Ad5 Hexon Protein Enhances the Protective Efficacy of a Protein Based Malaria Vaccine

A malaria vaccine is a public health priority. In order to produce an effective vaccine, a multistage approach targeting both the blood and the liver stage infection is desirable. The vaccine candidates also need to induce balanced immune responses including antibodies, CD4+ and CD8+ T cells. Protein-based subunit vaccines like RTS,S are able to induce strong antibody response but poor cellular reactivity. Adenoviral vectors have been effective inducing protective CD8+ T cell responses in several models including malaria; nonetheless this vaccine platform exhibits a limited induction of humoral immune responses. Two approaches have been used to improve the humoral immunogenicity of recombinant adenovirus vectors, the use of heterologous prime-boost regimens with recombinant proteins or the genetic modification of the hypervariable regions (HVR) of the capsid protein hexon to express B cell epitopes of interest. In this study, we describe the development of capsid modified Ad5 vectors that express a promiscuous Plasmodium yoelii T helper epitope denominated PyT53 within the hexon HVR2 region. Several regimens were tested in mice to determine the relevance of the hexon modification in enhancing protective immune responses induced by the previously described protein-based multi-stage experimental vaccine PyCMP. A heterologous prime-boost immunization regime that combines a hexon modified vector with transgenic expression of PyCMP followed by protein immunizations resulted in the induction of robust antibody and cellular immune responses in comparison to a similar regimen that includes a vector with unmodified hexon. These differences in immunogenicity translated into a better protective efficacy against both the hepatic and red blood cell stages of P. yoelii. To our knowledge, this is the first time that a hexon modification is used to deliver a promiscuous T cell epitope. Our data support the use of such modification to enhance the immunogenicity and protective efficacy of adenoviral based malaria vaccines.     

Construction and characterization of adenovirus serotype 5 packaged by serotype 3 hexon

Adenovirus serotype 5 (Ad5) has great potential for gene therapy applications. A major limitation, however, is the host immune response against Ad5 infection that often prevents the readministration of Ad5 vectors. In this regard, the most abundant capsid protein, hexon, has been implicated as the major target for neutralizing antibodies. In this study, we sought to escape the host neutralization response against Ad5 via hexon replacement. We constructed a chimeric adenovirus vector, Ad5/H3, by replacing the Ad5 hexon gene with the hexon gene of Ad3. The chimeric viruses were successfully rescued in 293 cells. Compared to that for the control Ad5/H5, the growth rate of Ad5/H3 was significantly slower and the final yield was about 1 log order less. These data indicate that the Ad3 hexon can encapsidate the Ad5 genome, but with less efficiency than the Ad5 hexon. The gene transfer efficacy of Ad5/H3 in HeLa cells was also lower than that of Ad5/H5. Furthermore, we tested the host neutralization responses against the two viruses by using C57BL/6 mice. The neutralizing antibodies against Ad5/H3 and Ad5/H5 generated by the immunized mice did not cross-neutralize each other in the context of in vitro infection of HeLa cells. Preimmunization of C57BL/6 mice with one of the two types of viruses also did not prevent subsequent infection of the other type. These data suggest that replacing the Ad5 hexon with the Ad3 hexon can circumvent the host neutralization response to Ad5. This strategy may therefore be used to achieve the repeated administration of Ad5 in gene therapy applications.     

Decreased pre-existing Ad5 capsid and Ad35 neutralizing antibodies increase HIV-1 infection risk in the Step trial independent of vaccination

Background

The Step trial raised the possibility that uncircumcised men with pre-existing Ad5 neutralizing antibodies carried an increased risk of HIV infection after vaccination. Thus, understanding Ad seropositivity in humans is important to the development of an AIDS vaccine. Here, we analyze the impact of different Ad5-specific neutralizing antibodies on immune function and clinical outcome.

Methods and Findings

Ad seropositivity in the Step trial volunteers was analyzed using chimeric rAd5/35 vectors to characterize their specificity for Ad5 fiber and non-fiber external (capsid) proteins. Immune responses and HIV seropositivity were correlated with the specificity of Ad5-neutralizing antibodies. Neutralizing antibodies induced by the vaccine in Ad5 seronegative subjects were directed preferentially to Ad5 capsid proteins, although some fiber-neutralizing antibodies could be detected. Pre-vaccination Ad5 serostatus did not affect the capsid-directed response after three vaccinations. In contrast, anti-fiber antibody titers were significantly higher in volunteers who were Ad5 seropositive prior to vaccination. Those Ad5 seropositive subjects who generated anti-capsid responses showed a marked reduction in vaccine-induced CD8 responses. Unexpectedly, anti-vector immunity differed qualitatively in Ad5 seropositive participants who became HIV-1 infected compared to uninfected case controls; Ad5 seropositive participants who later acquired HIV had lower neutralizing antibodies to capsid. Moreover, Ad35 seropositivity was decreased in HIV-infected subjects compared with uninfected case controls, while seroprevalence for other serotypes including Ad14, Ad28 and Ad41 was similar in both groups.

Conclusions

Together, these findings suggest that the case subjects were less immunologically responsive prior to infection. Subjects infected during the Step trial had qualitative differences in immunity that increased their risk of HIV-1 infection independent of vaccination.     

Immunogenicity when utilizing adenovirus serotype 4 and 5 vaccines expressing circumsporozoite protein in naive and Ad5 immune mice

ABSTRACT: BACKGROUND: Induction of potent long lasting effector T cell responses against liver stage malaria antigens strongly correlates with protection from malaria. While Adenovirus serotype 5 (Ad5) based malaria vaccine platforms have the ability to induce potent effector T cell responses against transgenes, high rates of pre-existing Ad5 immunity in malaria endemic regions has prompted study of alternative Ad serotype based malaria vaccines as replacements for Ad5 based malaria vaccines. The research described in this article examines the utility of alternative serotype adenovirus serotype 4 (Ad4) expressing a sporozoite surface protein (circumsporozoite protein (CSP)) (Ad4-CSP) to induce immune responses against CSP. The immunogenicity of Ad4-CSP was also tested in homologous and heterologous prime boost vaccinations in both Ad5 naive and Ad5 immune backgrounds as compared to use of Ad5-CSP. RESULTS: In Ad5 naive animals, use of Ad4-CSP priming vaccinations followed by boosting with Ad5-CSP (Ad4-CSP/Ad5-CSP) maximally increased the numbers of CSP specific cytokine secreting cytotoxic T cells relative to repeated use of Ad5-CSP. The Ad4-CSP/Ad5-CSP regimen also induced equivalent levels of CSP specific cell killing as did homologous prime-boost vaccinations with Ad5-CSP, despite stimulating lower numbers of CSP specific cytotoxic T cells. Priming with Ad4-CSP followed by a homologous boost resulted in significantly less CSP specific humoral responses than any other vaccination regimen tested in Ad naive animals. In Ad5 immune animals, addition of Ad4-CSP in homologous or heterologous prime boost resulted in inductions of higher CSP specific responses than animals repeatedly vaccinated with Ad5-CSP alone. However, the observed responses were well below those observed in similarly treated Ad naive mice. CONCLUSIONS: While the Ad4-CSP/Ad5-CSP and Ad5-CSP/Ad5-CSP vaccination regimens resulted in equivalent CSP specific killing in Ad naive animals, Ad4-CSP/Ad5-CSP achieved this result with a lower percentage of CSP specific CD8+ T cells and a higher number of IFNgamma secreting cells, suggesting that the Ad4-CSP/Ad5-CSP vaccination regimen elicits more efficient cytotoxic T cells. In Ad5 immune animals use of Ad4-CSP improved CSP specific immune responses as compared to repeated use of Ad5-CSP, but could not achieve the levels of immunogenicity observed when the same vaccine regimens were used in Ad naive animals. These data indicate the existence of some level of immunological cross-reactivity between these two adenovirus subgroups. Based on these results, it is suggested that future studies should undertake similarly stringent analyses of alternative Ad serotypes to establish their effectiveness as replacements for Ad5.     

Improved glioblastoma treatment with Ad5/35 fiber chimeric conditionally replicating adenoviruses

Adenovirus type 5 (Ad5)-based vectors have been used in clinical trials for glioblastoma treatment, but the capacity of Ad5 to infect human glioma cells was questioned. Seeking to improve the adenovirus transduction, we tested four Ad5-based vectors differing only in their fiber gene on permanent and short-term cultures of glioblastoma cells. A wild-type fiber Ad5 vector (Ad5.Luc) was compared to an RGD integrin-binding motif-containing fiber adenovirus (AdlucRGD) and the two fiber chimeras Ad5/3 and Ad5/35, with vector binding redirected to the Ad3 or Ad35 receptor, respectively. Compared to Ad5, the transduction of the tested short-term glioblastoma cultures with the vector Ad5/35.Luc, AdlucRGD and Ad5/3.Luc was enhanced by approximately 72%, approximately 13% and approximately 2%, respectively. To limit adenovirus spread, we aimed to develop conditionally replicative Ad5/35 vectors by targeting the expression of the essential E1 and E4 genes; in addition, some vectors had the E1Delta24 deletion. We analyzed eleven promoters for their activity in glioblastoma cells and determined the specificity of eight replicative adenovirus vectors in vitro. We evaluated the most promising vectors with E1/E4 under the control of the GFAP/Ki67 or E2F-1/COX-2 promoters, and the native Ad5 or the chimeric Ad5/35 fiber for their antineoplastic activity in a subcutaneous and intracranial glioblastoma xenograft model. Animals treated with the Ad5/35-based vectors showed significantly smaller tumors and longer survival than those treated with the homologous Ad5 vectors; no significant toxicity was observed in the intracranial model. Our data suggest that Ad5/35-based vectors are promising tools for glioblastoma treatment.     

Differential Specificity and Immunogenicity of Adenovirus Type 5 Neutralizing Antibodies Elicited by Natural Infection or Immunization

A recent clinical trial of a T-cell-based AIDS vaccine delivered with recombinant adenovirus type 5 (rAd5) vectors showed no efficacy in lowering viral load and was associated with increased risk of human immunodeficiency virus type 1 (HIV-1) infection. Preexisting immunity to Ad5 in humans could therefore affect both immunogenicity and vaccine efficacy. We hypothesized that vaccine-induced immunity is differentially affected, depending on whether subjects were exposed to Ad5 by natural infection or by vaccination. Serum samples from vaccine trial subjects receiving a DNA/rAd5 AIDS vaccine with or without prior immunity to Ad5 were examined for the specificity of their Ad5 neutralizing antibodies and their effect on HIV-1 immune responses. Here, we report that rAd5 neutralizing antibodies were directed to different components of the virion, depending on whether they were elicited by natural infection or vaccination in HIV vaccine trial subjects. Neutralizing antibodies elicited by natural infection were directed largely to the Ad5 fiber, while exposure to rAd5 through vaccination elicited antibodies primarily to capsid proteins other than fiber. Notably, preexisting immunity to Ad5 fiber from natural infection significantly reduced the CD4 and CD8 cell responses to HIV Gag after DNA/rAd5 vaccination. The specificity of Ad5 neutralizing antibodies therefore differs depending on the route of exposure, and natural Ad5 infection compromises Ad5 vaccine-induced immunity to weak immunogens, such as HIV-1 Gag. These results have implications for future AIDS vaccine trials and the design of next-generation gene-based vaccine vectors.Recombinant adenovirus (rAd)-based vectors are currently under investigation in a variety of gene therapy and T-cell-based vaccine clinical trials. There are more than 370 such ongoing clinical trials for broad applications, including infectious diseases and cancer therapy (http://www.wiley.co.uk/genetherapy/clinical/). Based on supportive data from nonhuman primate studies, rAd-based vectors have been developed and tested in human clinical trials to deliver human immunodeficiency virus (HIV-1) gene products that stimulate HIV-specific immune responses. Preexisting immunity to Ad serotype 5 (Ad5), from which most vectors are derived, is common in humans. Though neutralizing antibodies to Ad5 may reduce the immunogenicity of Ad5-based vectors in animal models (16), their effect on immunity in subjects with previous Ad5 infection is poorly understood. In the STEP trial, which tested a Merck rAd5 vaccine encoding HIV-1 Gag, Pol, and Nef, vaccination failed to show protection, either by lowering viral load or by decreasing acquisition of infection (3, 9, 12, 21). Furthermore, the possibility was raised that subjects with preexisting neutralizing antibodies from natural Ad5 infection may have carried an increased risk of HIV infection after vaccination. Thus, understanding the nature and immune effects of Ad5 seropositivity in humans is important to the development of vaccines against AIDS and other diseases.Ad5 is a common cause of respiratory disease and an occasional cause of gastroenteritis in humans, and exposure before adolescence is common in human populations (19). Such exposure stimulates both innate and adaptive immune responses that generate neutralizing antibodies and virus-specific T-cell responses (6). These antibodies can also synergize with each other to achieve maximum viral neutralization (7, 22). The capsid protein specificity of Ad5 neutralizing antibodies has been reported for humans following administration of rAd5 gene therapy vectors for advanced liver or lung cancer (7, 10). However, results were presented solely for antibodies induced by administration of rAd5. One report has assessed Ad5 neutralizing antibodies with a healthy human population that was Ad5 seropositive from natural exposure to the virus (18). The median titer of the population was presented, but the frequency of protein-specific neutralizing antibody has not been defined for humans.Here we describe the first report of the natural frequency and effect on immunization of neutralizing antibodies specific for different Ad capsid proteins in human subjects. We address the fundamental mechanisms of how humans generate neutralizing antibodies to a common cold virus that is in widespread use as a vector for gene therapy and vaccines. Such mechanisms may also be applicable to other nonenveloped viruses, including adeno-associated viruses and other viruses containing multiple envelope surface proteins, like influenza. To analyze the contribution of anti-capsid antibodies to neutralization by different human serum samples, wild-type and chimeric vectors were utilized. For example, a rAd type 5 (rAd5) vector with a fiber derived from Ad35 fiber (rAd5 F35) can be used to analyze the anti-Ad5 capsid response independent of fiber. Conversely, a rAd35 vector with a fiber transposed from Ad5 can determine the specificity of neutralization mediated by the Ad5 fiber. Using these vectors, we have analyzed human serum samples from two HIV vaccine clinical trials, VRC 006 and HVTN 204, in which a single-dose rAd5 vaccine alone and a three-dose DNA prime/single dose rAd5 boost vaccine encoding HIV-1 Env A,B, and C; Gag; and Pol, respectively, were administered. Thus, we sought to characterize the specificity of rAd5 neutralizing antibodies in Ad5-immune subjects and to determine their effect on immune responses elicited by vaccination.     

Immunogenicity of heterologous recombinant adenovirus prime-boost vaccine regimens is enhanced by circumventing vector cross-reactivity

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations has led to the development of recombinant adenovirus (rAd) vectors derived from rare Ad serotypes as vaccine candidates for human immunodeficiency virus type 1 and other pathogens. Vaccine vectors have been constructed from Ad subgroup B, including rAd11 and rAd35, as well as from Ad subgroup D, including rAd49. However, the optimal combination of vectors for heterologous rAd prime-boost vaccine regimens and the extent of cross-reactive vector-specific neutralizing antibodies (NAbs) remain poorly defined. We have shown previously that the closely related vectors rAd11 and rAd35 elicited low levels of cross-reactive NAbs. Here we show that these cross-reactive NAbs correlated with substantial sequence homology in the hexon hypervariable regions (HVRs) and suppressed the immunogenicity of heterologous rAd prime-boost regimens. In contrast, vectors with lower hexon HVR homology, such as rAd35 and rAd49, did not elicit detectable cross-reactive vector-specific NAbs. Consistent with these findings, rAd35-rAd49 vaccine regimens proved more immunogenic than both rAd35-rAd5 and rAd35-rAd11 regimens in mice with anti-Ad5 immunity. These data suggest that optimal heterologous rAd prime-boost regimens should include two vectors that are both rare in human populations to circumvent preexisting antivector immunity as well as sufficiently immunologically distinct to avoid cross-reactive antivector immunity.     

Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D

Recombinant adenovirus serotype 5 (rAd5) vector-based vaccines are currently being developed for both human immunodeficiency virus type 1 and other pathogens. The potential limitations associated with rAd5 vectors, however, have led to the construction of novel rAd vectors derived from rare Ad serotypes. Several rare serotype rAd vectors have already been described, but a detailed comparison of multiple rAd vectors from subgroups B and D has not previously been reported. Such a comparison is critical for selecting optimal rAd vectors for advancement into clinical trials. Here we describe the construction of three novel rAd vector systems from Ad26, Ad48, and Ad50. We report comparative seroprevalence and immunogenicity studies involving rAd11, rAd35, and rAd50 vectors from subgroup B; rAd26, rAd48, and rAd49 vectors from subgroup D; and rAd5 vectors from subgroup C. All six rAd vectors from subgroups B and D exhibited low seroprevalence in a cohort of 200 individuals from sub-Saharan Africa, and they elicited Gag-specific cellular immune responses in mice both with and without preexisting anti-Ad5 immunity. The rAd vectors from subgroup D were also evaluated using rhesus monkeys and were shown to be immunogenic after a single injection. The rAd26 vectors proved the most immunogenic among the rare serotype rAd vectors studied, although all rare serotype rAd vectors were still less potent than rAd5 vectors in the absence of anti-Ad5 immunity. These studies substantially expand the portfolio of rare serotype rAd vectors that may prove useful as vaccine vectors for the developing world.