Adenoviruses perform well as vaccine vectors for the delivery of a variety of viral, bacterial, and parasitic antigens (20
). rAd5 vectors in particular generate potent antigen-specific immune responses in mice, nonhuman primates, and humans, as we have observed when EBOV-GP is the target antigen. However, preclinical and human clinical studies have suggested that the potency of rAd5-based vectors may be compromised in individuals who have been exposed previously to Ad5 if they have a high level of immunity against the vector. The aim of the studies herein was to identify rAd vectors that can deliver the EBOV GP antigen in both Ad5-naïve and Ad5-immune subjects.
Since preexisting immunity against any viral vector has the potential to limit its effectiveness, we focused our attention on viruses that infect humans relatively rarely, as indicated by the prevalence of seropositive subjects and/or the low levels of neutralizing antibodies. Vesicular stomatitis virus (VSV) has been used successfully as a vector for filovirus vaccines, and antibody frequency against the vector in human subjects is presumed to be low, since it is primarily a veterinary pathogen and rarely causes symptomatic clinical infection in humans. However, the discovery of Chandipura virus as the causative agent of febrile illness and encephalitis in humans (35
) as well as documented human VSV exposure coincident with livestock outbreaks in the United States (37
) suggest that natural exposure may occur more frequently than has been assumed, but surveys have not been performed to evaluate human seroprevalence. In contrast, extensive sampling has been undertaken to determine the frequency of anti-adenovirus antibodies in human sera (1
). The adenovirus serotypes Ad35 and Ad26 were selected for vaccine development in the current work due to their low seroprevalence in humans.
rAd35-GP vaccination of macaques generated antigen-specific antibody and T-cell responses in individual subjects within the range observed previously when rAd5 was used as the delivery vector. The average anti-GP antibody titer for all rAd35 vaccinees (irrespective of dose), 1:1,400, was lower than the average for all historical subjects vaccinated with 1010 rAd5-GP (1:11,000, n = 17), providing an initial indication that vector potency might differ between the two serotypes if antibody titer is an immune correlate of protection for rAd35 as it is for rAd5 EBOV vaccines. CD4+ and CD8+ T-cell responses were detectable in most subjects prior to infectious challenge, though the absolute magnitude cannot be compared to that for rAd5 vaccinees not included in these studies in the absence of PBMC samples for assay bridging controls.
Vaccination with rAd35 vectors effectively induced antigen-specific antibody and T-lymphocyte immune responses in both rAd5-naïve or rAd5-immune subjects, suggesting that rare serotype vector genomes are sufficiently distant from common serotypes to resist heterologous vector-directed immunity. This feature of vector performance will be important to circumvent preexisting immunity stemming not only from natural viral infection but also from the use of heterologous vectors in priming immunizations or vaccination against other pathogens. Indeed, rAd35-GP inoculation provided a potent boost of both cell-mediated and antibody responses in macaques primed with rAd26-GP. This result was intriguing, since it demonstrates a clear difference in vector potency for the induction of secondary versus primary immune responses; the ability of rAd35-GP to boost the immune response was not predicted by the magnitude of responses observed after the priming immunization. These data may indicate that rAd35 and other rAd vectors have a higher transduction efficiency in certain populations or activation states of target dendritic cells, as suggested recently by Lindsay et al. (23
), which may be, in this case, more abundant or accessible during secondary immune responses.
The rAd26/rAd35 prime-boost vaccine provided uniform protection, and rAd26 proved to be more potent than rAd35 as a single-shot vaccine against EBOV infection, mediating survival in up to 75% of vaccinated macaques at the highest dose tested. rAd26-GP vaccines demonstrated a clear dose response for the induction of protective immunity, suggesting that marginal improvements in antigen expression could increase the potency of rAd26-based vaccines to generate uniform protection against high doses of EBOV challenge, such as those used herein. Interestingly, the high degree of protection offered by rAd26-GP vectors compared to rAd35-GP at a matched dose (1011 particles) was associated with higher ELISA anti-GP titers, 1:4,500 versus 1:1,400, respectively. These data raise the possibility that prechallenge antibody titers could serve as an immune correlate of protection against ZEBOV infection for the Ad26/Ad35 combination in addition to within-vector groups, as has been observed for rAd5-GP vaccines. The order of potency for induction of antibody responses predicted the rank order for protection across vector groups.
The studies herein tested vaccine vectors by comparing them singly and in combination, demonstrating the utility of alternative-serotype rAd for use as vaccine vectors in primates. rAd26 and rAd35 were less potent vectors than rAd5 or VSV (11
) when used as single-modality vaccines, but they performed well when combined, mediating full protection in macaques. This promising result in macaques suggests that these vaccines will be immunogenic in humans for the induction of anti-EBOV responses. Although it is not yet known whether the protective doses used here will be tolerated in humans, an rAd26-based HIV vaccine was tolerated at similar doses in a recent human clinical trial (3
). The nonhuman primate studies here suggest that these vaccines may be most useful in a prime-boost combination. By administering rAd26 with Ad35 in a heterologous vaccination regimen, humoral and cellular immune responses were efficiently increased. Because of the high magnitude of antigen-specific responses achieved by heterologous prime-boost, it has been proposed that long-term immunity may be optimally achieved by priming rAd with DNA (38
). Since DNA requires multiple primes and does not induce rapid protection like rAd vectors do, heterologous rAd prime-boost may provide an optimal opportunity to generate a balance between the inductions of rapid and long-lasting protective immunity.