For reasons that are not clear, there are large differences in the ability to induce CD8+ T-cells by diverse viral and non viral genetic vaccine carriers. This is particularly true in humans where many different approaches have failed to induce CD8+ T cell immunity even in the face of positive results obtained in pre-clinical animal studies.Ad vectors derived from strains that have not circulated widely in humans (“rare serotypes”) are under investigation as vaccine vectors, based on the expectation that they would be as potent as Ad5 while being insensitive to neutralisation by anti-Ad5 antibodies.
To help predicting the immunological potency in humans of the different Ad vectors, we screened them for their ability to stimulate an immune response over a wide range of doses using the clinically validated human Ad5 as a benchmark. By dose/response comparative studies in mice and non-human primates, we found that Ad vectors from the rare human serotypes Ad24, Ad26, Ad34 and Ad35 are substantially less immunogenic (100-1000 fold) than the clinically validated Ad5. Interestingly, Ad6 was comparable to Ad5 in these experiments, a finding consistent with previous immunological data obtained in mice, non-human primates and humans (4
), and an important validation of the dose/response approach to establish the relative immunological potency of Ad vectors across species.
The association between low seroprevalence and weak immunological potency may not be a mere coincidence, but rather reflect a reduced fitness of the virus in the human population possibly leading to a reduced immunological potency. Thus, while potentially overcoming the problem of the pre-existing immunity to Ad5, the use of Ad vectors from rare human serotypes appears not to address other issues such as the immunological potency, thus running a high risk of failure in humans, as suggested by recent Phase I clinical data with an Ad35-based HIV candidate vaccine (33
). Preliminary flow cytometry data from this trial show rather low frequency of responders with 8% and 14% responding subjects over total examined for CD8 and CD4, respectively.
Lacking a useful biomarker of Ad vector immunogenicity in vivo
, our strategy to identify alternative Ad vectors with all the features necessary for vaccine development was to generate a large collection of such vectors and to screen them to find suitable candidates for vaccine development. This ‘reverse vectorology’ approach required a “library” of different Ads to be available for the screening. To generate such a library we decided to isolate novel Ad strains from chimpanzees. This choice was based on the prediction that the substantial genetic diversity between individuals of the Pan species would extend to their pathogens (34
) making it possible to isolate a large repertoire of different Ads. This turned out to be the case as we isolated a large number of different Ads from chimpanzees and bonobos which were classified into the same groups as human Ads based on the amino acid sequence of their hexon proteins. Among these isolates we could identify at least twenty-five different strains based on hexon homology and we confirmed that they represent individual serotypes by in vitro
cross-neutralization assays and in vivo
Sequencing the whole genome of some of the ChAd isolates (ChAd3, ChAd6, ChAd9, ChAd19, ChAd43, ChAd63, ChAd83, PanAd1, PanAd2, PanAd3) revealed that they are strongly related to human Ads, showing a high degree of DNA homology with human Ads belonging to the same species (80-95%) and similar genomic structure. The latter feature was particularly relevant to map the ChAd E1 regions to be deleted for the generation of replication incompetent vectors. We could not identify a feature in the Chimpanzee adenovirus DNA sequence that would allow to distinguish between viruses of human or chimpanzee origin.
The ChAd collection was used as a source of vectors for functional screening to assess: i) growth capability in HEK293 and PER.C6 cells, ii) immunological potency in mice and non-human primates, iii) sensitivity to neutralizing antibodies present in humans.
Replication defective E1-deleted ChAd vectors were efficiently propagated in PER.C6 and HEK293 cells, confirming the functional equivalence of human Ad5 and chimpanzee Ad E1. Importantly, we did not observe the formation of RCA during propagation of ChAd in HEK293, indicating that there is insufficient sequence homology between human and chimpanzee Ads to allow for homologous recombination between the two genomes, and opening the possibility to use HEK293 for large scale manufacturing of ChAds.
All ChAds revealed significant diversity in the hypervariable regions of the hexon protein from the highly seroprevalent Ad5 and were not neutralized by anti-Ad5 antibodies in vitro and in vivo. Consequently, they were all found to be very rarely neutralized by antibodies present in humans, and for about half of them none of the tested sera displayed any neutralizing activity. Conversely, most ChAds were very often neutralized by chimpanzee sera (data not shown), further supporting the notion that serologically distinct strains of Adenoviruses circulate in the two species and that Adenovirus infection across human and chimpanzees is a very rare event.
Screening for immunological potency by dose/response in mice confirmed that there is a high degree of heterogeneity between ChAds as already observed for human Ads. Nevertheless, given the large number of screened candidates we were able to identify some ChAds with immunological potency equivalent to Ad5 (ChAd3, ChAd63, ChAd83, PanAd1, PanAd2 and PanAd3), and several others with a slightly lower immunogenicity within a factor of five to ten-fold. Importantly, the high level of immunogenicity of the top ranking ChAd3 and PanAd3 was confirmed in non-human primates, where they induced a level of T-cell response comparable to that of Ad5 even at low dose (108
vp). Similarly, we recently showed that another high scoring ChAd vector (ChAd63) encoding for the Malaria TRAP antigen induced a very potent T-cell response in Rhesus macaques (35
). Notably, the ability of ChAds to induce strong cellular immunity is not an antigen-dependent phenomenon as we could induce substantial T-cell response by immunising rodents and non human primates with ChAd vectors encoding different antigens from infectious agents such as HIV, HCV, Malaria, HSV2, Ebola, RSV and Influenza as well as cancer.
Despite their overall similarity in structure and genome organization, human and chimpanzee Adenoviruses display a wide range of immunological potency when used as replication defective vectors in vivo. We were unable to identify a mechanism for such a large difference in the immunological potency between Adenovirus vectors. Nevertheless we could establish a correlation between the efficiency of stimulating the adaptive immune system and the phylogenetic classification into the different species according to the homology of the hexon protein sequence. In fact, all tested group C viruses (both human and chimpanzee) were very potent with a breakpoint of 107 vp or lower, while human or chimpanzee group B Ad were immunogenic only when used at the dose of 109 vp or higher. Human group D and some of the group E ChAd viruses were also rather weak carriers as they were able to induce an immune response only at the dose of 108 vp. Within the group E ChAd we could distinguish two clusters on the basis of their hexon sequence homology: ChAd6, 9, 10, 43, 47, 63, 83 and 147 belonged to one family (E1) and all the others clustered into a separate family (E2). Interestingly, most of the E1 viruses were potent vectors, while members of the E2 family displayed lower immunogenicity.
One of the possible explanations for the different immunological potency displayed by human and chimpanzee Ad strains could be their receptor usage and/or cell tropism (36
). Indeed, the most potent group C and group E Adenoviruses use the CAR receptor to infect a variety of different cell types, while Group B Adenoviruses (ie.: the human Ad11, Ad34 and Ad35 and the chimpanzee ChAd30) recognise the CD46 surface protein and infect DC cells more efficiently than group C isolates in vitro
). This finding originally led to the hypothesis that the latter viruses would be a more effective class of genetic vaccine carrier. In contrast, we showed that they are the least potent among all human and chimpanzee derived Ad vectors. To explain this apparent contradiction one might speculate that highly efficient infection of professional antigen presenting cells by the group B Ad would lead to a more rapid elimination of the vector thereby reducing the efficiency and longevity of expression of the encoded antigen. This hypothesis is consistent with the low prevalence of group B Adenoviruses in both humans and chimpanzees which may result from a more efficient immunological clearance as compared to other Adenoviruses such as those from the group C or E.
Predicting the immunological potency in humans is a key factor for vaccine vector development. Final validation of our dose/response immunological screening in mice came from the observation that two of the most potent ChAd vectors, ChAd3 and ChAd63, induced T-cell immunity in 100% of immunised human volunteers with average peak responses exceeding 1500 IFNγ SFC/106 PBMCs for ChAd3 and about 1000 IFNγ SFC/106 PBMCs for ChAd63. These are by far the highest levels of T-cell responses ever observed in humans with a single non-replicating genetic vaccine vector. Of note, the ChAd-induced cellular response was characterised by extremely potent IFNγ+ CD8 T-cells in all tested species from rodents to humans, with high values of over 1% antigen-specific IFNγ+ CD8 T-cells in ChAd3 vaccinated healthy humans.
The success of vaccine-induced T-cell immunity in clearing infected cells before the onset of an acute disease or the establishment of a chronic infection is likely to depend on two factors: i) the level of CD8+ T cell effectors that are present at the time of the infection and can rapidly recognise and eliminate infected cells, and ii) the presence of a pool of memory population of T cells that are capable of rapid re-expansion upon encountering of the pathogen antigens. Our finding that non-human primates immunised with ChAd3 developed HIV gag-specific IFNγ+ T-cells which persisted for more than five years as detected by ex-vivo ELISpot indicates that ChAd have the potential to induce high frequencies of long lasting antigen-specific T-cells with effector function. Furthermore, these long lived T-cell pools also displayed the typical feature of a memory population as they underwent rapid expansion in vivo upon boosting with a second non cross-reacting ChAd vector encoding the same HIV gag antigen.
We could demonstrate that pre-immunization with high doses of a heterologous ChAd did not affect the efficiency of immunization of a subsequently injected ChAd from a different serotype. Thus, our collection of potent, non cross-reacting ChAd vectors can be exploited for several vaccine applications following the strategy of ‘one vector-one disease’ to avoid potential interference between different vaccines. Additional evidence in favour of this strategy comes from experiments in non-human primates where we have successfully immunised animals with ChAd3-gag or ChAd3-NSmut and subsequently with ChAd63 encoding the AMA-1 Malaria antigen without any detectable interference between the different vaccine vectors. In addition, the availability of several different ChAd vectors allows the design of a vaccination strategy based on heterologous prime-boost modality with serologically distinct ChAd to maximize immunization strength.