The Ad4-H5-Vtn vector vaccine was well tolerated; symptoms of reactogenicity were generally mild and resolved within 7 days of vaccination. No serious treatment-related adverse events occurred. Ad4-H5-Vtn was not detected by PCR analysis of samples from household contacts of participants. After three vaccinations, haemagglutinin-specific antibody responses to all doses were scarce and of low titres, with HAI seroconversion ranging from 4% to 19% among the vaccine cohorts, compared with 7% among placebo recipients. However, after one inactivated parenteral H5N1 immunisation, HAI seroconversion occurred in 80% of participants (100% in the highest-dose cohort), compared with 36% of placebo recipients, and GMT was significantly higher in participants who had been primed with Ad4-H5-Vtn than in those who had received placebo (panel
Panel. Research in context
We searched the medical literature (PubMed, literature cited in relevant publications) for clinical studies of replication-competent vector vaccines, avian influenza vaccines, and Ad4 or Ad7 vaccines. The PubMed search used the terms “pandemic influenza”, “replication competent vector”, “adenovirus 4”, and “vector vaccine”; it was not limited by date or language. The date of the last search was Feb 21, 2012. We noted that few replicating vector vaccines have progressed to clinical assessment, and that most, while perhaps priming and inducing a cellular immune response to transgene expressed antigens, did not induce robust antibody responses. We noted that H5N1 vaccines, either inactivated or live attenuated, generally did not induce robust antibody responses compared with influenza vaccines, either seasonal or other avian influenza strains, such as H9N2 or H7N3.
As far as we are aware, our study is the first clinical trial of a replication-competent Ad4 vector vaccine for any indication, and one of the few replication-competent vector clinical studies for influenza. We confirmed that the orally administered Ad4 H5N1 vector vaccine seemed to have a safety profile, including low transmissibility, similar to the parent Ad4 vaccine currently used by the US military. Similar to other vector vaccines, the Ad4 H5N1 vector induced a cellular immune response but only minimum antibody response. However, one boost with inactivated H5N1 vaccine led to robust haemagglutination inhibition and H5N1 neutralising antibody responses. This study provides evidence supporting the oral replicating Ad4 vector prime-inactivated boost as a vaccine approach for pandemic influenza, and potentially other infections for which both cellular and antibody responses are needed. However, confirmatory studies will be needed to better assess doses and intervals between prime and boost.
The Ad4-H5-Vtn vector was bioengineered by partial deletion in the E3 region and insertion of the H5 haemagglutinin gene. The function of the E3 region in Ad4 is unknown, but, on the basis of strong E3 homology with Ad5, it is thought to be important in modulation of host-antiviral responses.22
Since Ad4 does not replicate in non-human animals except chimpanzees,23
replication was assessed in human cell lines before we made an investigational new drug application and was shown to be attenuated compared with unmodified Ad4.12
Nevertheless, the recorded occurrence of Ad4 seroconversion and GMTs in both Ad4 seronegative and seropositive Ad4-H5-Vtn recipients with doses of 109
) are much the same or higher than in the US military Ad4/Ad7 vaccine phase 1 study.13
At these high doses, Ad4-H5-Vtn induced haemagglutinin-specific cellular responses and primed for HAI antibody responses, even in participants with pre-existing Ad4 immunity.
The Ad4-H5-Vtn vaccine also resembled the US military Ad4 vaccine with respect to safety and a low rate of transmissibility.11,24–26
Although asymptomatic transmission to intimate household contacts and children had been noted in studies before licensure, the US military Ad4 vaccine did not seem to be transmitted among military recruits.11,24–26
In our study of Ad4-H5-Vtn, no dose limiting or notable toxic effects were identified with doses ranging from 107
VP to 1011
VP. All household contacts were enrolled into the study and monitored for potential acquisition of Ad4-H5-Vtn infection. No confirmed transmission to these contacts occurred, although asymptomatic seroconversion to Ad4 was recorded in two contacts of vaccinees and one contact of a placebo recipient. These were presumed seroconversions to wild-type Ad4, since Ad4-H5-Vtn was not detected in throat or rectal swabs of these three household contacts, but seroconversion attributable to Ad4-H5-Vtn infection cannot be ruled out in the household contacts of the two vaccinees. Transmission to contacts of vaccinees is always a potential concern for live vaccines. Although the safety record of the US military Ad4 and Ad7 vaccines, including safety in contacts of vaccinees,11,24–26
and the absence of confirmed transmission in our study provide reassurance, assessment of transmissibility to, and safety in, vulnerable contacts will require further studies.
The Ad4-H5-Vtn vaccine induced haemagglutinin-specific cellular immune responses but only primed for haemagglutinin-specific antibody responses. However, after one inactivated H5N1 parenteral vaccine boost, we noted substantial HAI and neutralising antibody responses with occurrence of seroconversion and GMTs higher than achieved with unadjuvanted, inactivated H5N1 vaccines.14,27
For example in another study,14
after two doses of 90 μg inactivated H5N1 subvirion vaccine only 58% of participants achieved the prespecified protective HAI titre of 1/40 or more and 57% seroconverted. By contrast, we showed that after priming with Ad4-H5-Vtn and boosting with inactivated H5N1 vaccine, post-boost seroprotection and seroconversion were 80% and 80% with 1010
VP and 89% and 100% with 1011
VP. These proportions are higher28
to seroprotection rates for adjuvanted H5N1 vaccines or DNA priming followed by inactivated H5N1 boosting.32
In the DNA priming study,32
post-boost antibody responses were much the same, irrespective of whether vaccinees received one or two priming doses. Although the H5N1 boost was given mostly to volunteers who had received all three doses of the oral Ad4-H5-Vtn, the pattern of cellular immune response and vaccine take, both occurring predominantly after the first Ad4-H5-Vtn administration, suggest that one oral dose would have been sufficient for priming. A phase 2 study to confirm that one dose of Ad4-H5-Vtn is sufficient to prime for robust post-boost antibody responses, to investigate dosing of boost vaccine, and to assess the potential for broadening of response by cross-clade boosting, is being planned. Although speculation about the relative advantages and disadvantages of the oral Ad4 replicating vector approach versus DNA or replication-defective vectors might be premature, potential advantages, such as ease and reduced costs both of storage and of self-administration for an oral vaccine formulated as a capsule or tablet might be important in a national or worldwide response to an avian influenza pandemic. Though safety concerns about replicating vectors exist, the safety of the parent Ad4 vaccine virus, well established over many years by the US military,11
provides some reassurance.
The profile of immune response to the oral Ad4-H5-Vtn prime—ie, low occurrence of HAI or neutralising antibody responses, but significant H5 haemagglutinin-specific cellular responses—resembles responses to live-attenuated H5N1, H5N2, and H6N1 intranasal candidate vaccines.33–35
We do not know whether, in the absence of haemagglutinin-protein boosting, the predominantly cellular immune response induced by Ad4-H5-Vtn, or a rapid anamnestic antibody response on exposure to H5N1, would be sufficient to protect against H5N1 infection or disease. The correlates of protection for live-attenuated seasonal influenza vaccine are not well understood, particularly in children, and some reports suggest protection is mediated by local mucosal, cellular immune responses, or both, and that antibody titres much higher than the conventional HAI titre of at least 1/40 are required.36–38
Thus, we do not know whether one or several doses of the Ad4-H5-Vtn would be protective without parenteral boost, or whether the antibody response engendered by a prime-boost approach would be protective. Nevertheless, on the basis of our results, we might be able to infer that for pandemic H5N1 influenza, one oral Ad4-H5 priming dose, followed by a parenteral boost, would give better antibody response rates than two doses of inactivated vaccine, and, unlike the conventional parenteral vaccines, induce a robust cellular immune response. Potential benefits, such as a reduction of the number of doses of inactivated pandemic influenza vaccine required to be manufactured in a short time, and possibly prepandemic priming, have been suggested for the prime-boost approach.39
A possible reason why replicating Ad4-H5-Vtn by itself did not induce a good haemagglutinin-specific antibody response is that the H5 haemagglutinin antigen intrinsically is a poor immunogen, and that an Ad4 vector with a different transgene encoded antigen might have induced robust antibody responses without boost. Experience with other avian influenza vaccines is consistent with poor intrinsic immunogenicity of the H5 haemagglutinin. Two dose regimens of live-attenuated versions of H5N1 or H5N2 induced HAI antibody responses in only 10%34
of volunteers, whereas analogous live-attenuated avian influenza H9N2 and H7N3 vaccines had responses as high as 92% and 62%, respectively.40,41
Cell tropism attributable to inherent or unique properties of the Ad4 vector virus remains another possible explanation. However, in chimpanzees in which Ad4 vectors replicate to a limited extent, they have induced good HIV-Env antibody responses,42
and in other animals, including non-human primates, in which Ad4 vectors perform as a replication-defective vector, robust HIV-Env antibody or HAI responses have also been induced.12,43
Another possible explanation is that replicating vectors might inherently express their transgenes in a manner not conducive to direct stimulation of antibody. Analogous results (ie, marginal antibody response to the transgene encoded antigen but significant priming for subsequent boost) were noted with a replication-competent vaccinia vector expressing HIV gp160,7,8
but safety issues with vaccinia have precluded its wide development.44
Other replicating viral vectors have induced cellular responses, but only poor antibody responses in clinical trials.6
Several potential explanations exist for the low haemagglutinin-specific binding antibody and sporadic HAI responses, despite haemagglutinin-specific cellular responses. Oral administration of the Ad4-H5-Vtn vector efficiently primed for HAI antibody responses as evidenced by the robust antibody responses to boosting with inactivated parenteral H5N1 vaccine. Since heamagglutinin-specific cellular responses were induced, which would presumably provide helper functions, and low level haemagglutinin-specific binding antibody was also generated, it is likely that sufficient antigen might not have been available for robust B-cell activation and maturation. Insufficient haemagglutinin antigen presentation might have several causes including insufficient amounts of expression, suboptimum conformation (which would have a lesser effect on T-cell responses), lack of transport of expressed antigen to locations available for sampling by antigen-presenting cells and B cells, or degradation of the antigen. Further human experience with these Ad4 recombinant vectors expressing either Bacillus anthracis protective antigen, HIV-1 mosiac gag, or HIV-1 envelope will come from phase 1 trials in 2013, and the results might show whether immune profiles noted in this trial were the result of the characteristics of the H5 antigen or inherent properties of the oral administration of these replication-competent vectors.
Irrespective of the cause for the poor antibody response after oral administration of the Ad4-H5-Vtn vaccine, its immunogenicity—vector induction of a cellular immune response but with antibody response only after heterologous protein boosting—is the profile that was noted after immunisation with a canarypox vector and rEnv boost in the only HIV vaccine trial to have shown any, if only modest, efficacy.45
A vaccination regimen consisting of priming by a replicating Ad4 HIV-Env vector, followed by rEnv parenteral boosting might provide a similar or better immunological profile, particularly if oral priming by adenovirus leads to a mucosal cellular response as shown in animals.1,46,47
Replication-competent vector vaccines have, for many years, been proposed as potentially being the best avenue to a successful HIV-1 vaccine, since they are deemed to approximate more closely the features of live-attenuated vaccines, induce HIV-specific immune responses at the virus mucosal entry point, and might have a stronger immunogenicity than non-replicating vectors by induction of more potent innate and adaptive immune responses.2
Ad4 vaccine vectors expressing HIV-1 Env and Gag, to be followed by rEnv boost are currently scheduled for phase 1 clinical trials in 2013. Replicating Ad4 vector vaccines might also be effective in prime-boost regimens for other infections such as malaria, herpes simplex virus, and cytomegalovirus, for which both cellular and antibody responses seem to be needed for protection.