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Intramuscular delivery of human adenovirus (HAdV)-based vaccines leads to rapid recruitment of neutrophils, which then release antimicrobial peptides/proteins (AMPs). How these AMPs influence vaccine efficacy over the subsequent 24 h is poorly understood. In this study, we asked if human neutrophil protein 1 (HNP-1), an α-defensin that influences direct and indirect innate immune responses to a range of pathogens, impacts the response of human phagocytes to three HAdV species/types (HAdV-C5, -D26, -B35). We show that HNP-1 binds to the capsids and redirects HAdV-C5, -D26, and -B35 to Toll-like receptor 4 (TLR4), which leads to internalization, an NLRP3-mediated inflammasome response, and interleukin 1 beta (IL-1β) release. Surprisingly, IL-1β release was not associated with notable disruption of plasma membrane integrity. These data further our understanding of HAdV vaccine immunogenicity and may provide pathways to extend the efficacy. IMPORTANCE This study examines the interactions between danger-associated molecular patterns and human adenoviruses, and their impact on vaccines. HAdVs and HNP-1 can interact, and these interactions will modify the response of antigen-presenting cells, which will influence vaccine efficacy.

The global COVID-19 pandemic has sparked intense interest in the rapid development of vaccines as well as animal models to evaluate vaccine candidates and to define immune correlates of protection. We recently reported a mouse-adapted SARS-CoV-2 virus strain (MA10) with the potential to infect wild-type laboratory mice, driving high levels of viral replication in respiratory tract tissues as well as severe clinical and respiratory symptoms, aspects of COVID-19 disease in humans that are important to capture in model systems. We evaluated the immunogenicity and protective efficacy of novel rhesus adenovirus serotype 52 (RhAd52) vaccines against MA10 challenge in mice. Baseline seroprevalence is lower for rhesus adenovirus vectors than for human or chimpanzee adenovirus vectors, making these vectors attractive candidates for vaccine development. We observed that RhAd52 vaccines elicited robust binding and neutralizing antibody titers, which inversely correlated with viral replication after challenge. These data support the development of RhAd52 vaccines and the use of the MA10 challenge virus to screen novel vaccine candidates and to study the immunologic mechanisms that underscore protection from SARS-CoV-2 challenge in wild-type mice. IMPORTANCE We have developed a series of SARS-CoV-2 vaccines using rhesus adenovirus serotype 52 (RhAd52) vectors, which exhibit a lower seroprevalence than human and chimpanzee vectors, supporting their development as novel vaccine vectors or as an alternative adenovirus (Ad) vector for boosting. We sought to test these vaccines using a recently reported mouse-adapted SARS-CoV-2 (MA10) virus to (i) evaluate the protective efficacy of RhAd52 vaccines and (ii) further characterize this mouse-adapted challenge model and probe immune correlates of protection. We demonstrate that RhAd52 vaccines elicit robust SARS-CoV-2-specific antibody responses and protect against clinical disease and viral replication in the lungs. Further, binding and neutralizing antibody titers correlated with protective efficacy. These data validate the MA10 mouse model as a useful tool to screen and study novel vaccine candidates, as well as the development of RhAd52 vaccines for COVID-19.

Adenovirus (AdV)-based vectors are popular experimental vaccine vectors, but despite their ability to induce strong immune responses, their application is impeded by widespread preexisting immunity against many AdV types that can impair or even abrogate the induction of transgene-specific immune responses. Therefore, the development of vectors based on AdV types with a low seroprevalence is important for effective AdV-based immunization in humans. We investigated the immunization efficacy of vectors based on AdV type 48 (Ad48) and Ad50 in the ovalbumin (ova) model as well as the Friend retrovirus (FV) model, which allows testing of the protective effect of vaccine-induced immunity. Using ova-encoding vectors, we found a significantly lower induction of ova-specific CD8 + T cells and antibody responses by Ad48- and Ad50-based vectors than by Ad5-based vectors. Similarly, we found a reduced induction of FV-specific CD8 + T cell responses in Ad48- and Ad50.Leader-Gag-immunized mice compared with that in Ad5-immunized mice; however, some of those mice were able to control the FV infection, and protection correlated with the level of neutralizing antibodies 10 days after FV challenge. Analyses of the AdV-specific antibodies and CD8 + T cells induced by the individual AdV types revealed a high level of cross-reactivity, and the efficacy of Ad48-based immunization was impaired in Ad5-preimmune mice. Our results show that the immunity induced by Ad48- and Ad50-based vectors is reduced compared to that induced by Ad5 and is sufficient to control FV infection in only some of the immunized mice. A high level of cross-reactivity suggests that AdV preimmunity must be considered even when applying rare AdV-based vectors. IMPORTANCE AdV-based vectors are important tools for the development of vaccines against a wide range of pathogens. While AdV vectors are generally considered safe and highly effective, their application can be severely impaired by preexisting immunity due to the widespread seroprevalence of some AdV types. The characterization of different AdV types with regard to immunogenicity and efficacy in challenge models is of great importance for the development of improved AdV-based vectors that allow for efficient immunization despite anti-AdV immunity. We show that the immunity induced by an Ad48-based vector is inferior to that induced by an Ad5-based vector but can still mediate the control of an FV infection in highly FV-susceptible mice. However, the efficacy of Ad48-based immunization was impaired in Ad5-preimmune mice. Importantly, we found cross-reactivity of both the humoral and cellular immune responses raised by the individual AdV types, suggesting that switching to a different AdV type may not be sufficient to circumvent preexisting anti-AdV immunity., (Copyright © 2019 American Society for Microbiology.)

Since the 1970s, replication-competent human adenoviruses 4 and 7 have been used as oral vaccines to protect U.S. soldiers against the severe respiratory diseases caused by these viruses. These vaccines are thought to establish a digestive tract infection conferring protection against respiratory challenge through antibodies. The success of these vaccines makes replication-competent adenoviruses attractive candidates for use as oral vaccine vectors. However, the inability of human adenoviruses to replicate efficiently in laboratory animals has hampered the study of such vectors. Here, we used mouse adenovirus type 1 (MAV-1) in mice to study oral replication-competent adenovirus-based vaccines. We show that MAV-1 oral administration provides protection that recapitulates the protection against homologous respiratory challenge observed with adenovirus 4 and 7 vaccines. Moreover, live oral MAV-1 vaccine better protected against a respiratory challenge than inactivated vaccines. This protection was linked not only with the presence of MAV-1-specific antibodies but also with a better recruitment of effector CD8 T cells. However, unexpectedly, we found that such oral replication-competent vaccine systemically spread all over the body. Our results therefore support the use of MAV-1 to study replication-competent oral adenovirus-based vaccines but also highlight the fact that those vaccines can disseminate widely in the body. IMPORTANCE Replication-competent adenoviruses appear to be promising vectors for the development of oral vaccines in humans. However, the study and development of these vaccines suffer from the lack of any reliable animal model. In this study, mouse adenovirus type 1 was used to develop a small-animal model for oral replication-competent adenovirus vaccines. While this model reproduced in mice what is observed with human adenovirus oral vaccines, it also highlighted that oral immunization with such a replication-competent vaccine is associated with the systemic spread of the virus. This study is therefore of major importance for the future development of such vaccine platforms and their use in large human populations., (Copyright © 2019 American Society for Microbiology.)

Human and chimpanzee adenovirus vectors are being developed to circumvent preexisting antibodies against common adenovirus vectors such as Ad5. However, baseline immunity to these vectors still exists in human populations. Traditional cloning of new adenovirus vaccine vectors is a long and cumbersome process that takes 2 months or more and that requires rare unique restriction enzyme sites. Here we describe a novel, restriction enzyme-independent method for rapid cloning of new adenovirus vaccine vectors that reduces the total cloning procedure to 1 week. We developed 14 novel adenovirus vectors from rhesus monkeys that can be grown to high titers and that are immunogenic in mice. All vectors grouped with the unusual adenovirus species G and show extremely low seroprevalence in humans. Rapid cloning of novel adenovirus vectors is a promising approach for the development of new vector platforms. Rhesus adenovirus vectors may prove useful for clinical development. IMPORTANCE To overcome baseline immunity to human and chimpanzee adenovirus vectors, we developed 14 novel adenovirus vectors from rhesus monkeys. These vectors are immunogenic in mice and show extremely low seroprevalence in humans. Rhesus adenovirus vectors may prove useful for clinical development., (Copyright © 2018 Abbink et al.)

Adenovirus (Ad)-based immunization is a popular approach in vaccine development, and Ad-based vectors are renowned for their potential to induce strong CD8 + T cell responses to the encoded transgene. Surprisingly, we previously found in the mouse Friend retrovirus (FV) model that Ad-based immunization did not induce CD8 + T cell responses to the FV Leader-Gag-derived immunodominant epitope GagL 85-93 We show now that induction of GagL 85-93 -specific CD8 + T cells was highly effective when leader-Gag was delivered by plasmid DNA immunization, implying a role for Ad-derived epitopes in mediating unresponsiveness. By immunizing with DNA constructs encoding strings of GagL 85-93 and the two Ad-derived epitopes DNA-binding protein 418-426 (DBP 418-426 ) and hexon 486-494 , we confirmed that Ad epitopes prevent induction of GagL 85-93 -specific CD8 + T cells. Interestingly, while DBP 418-426 did not interfere with GagL 85-93 -specific CD8 + T cell induction, the H-2D d -restricted hexon 486-494 suppressed the CD8 + T cell response to the H-2D b -restricted GagL 85-93 strongly in H-2 b/d mice but not in H-2 b/b mice. This finding indicates that competition occurs at the level of responding CD8 + T cells, and we could indeed demonstrate that coimmunization with an interleukin 2 (IL-2)-encoding plasmid restored GagL 85-93 -specific CD8 + T cell responses to epitope strings in the presence of hexon 486-494 IL-2 codelivery did not restore GagL 85-93 responsiveness in Ad-based immunization, however, likely due to the presence of further epitopes in the Ad vector. Our findings show that seemingly immunodominant transgene epitopes can be dominated by Ad-derived epitopes. These findings underline the importance of thorough characterization of vaccine vectors, and modifications of vectors or immunogens may be required to prevent impaired transgene-specific immune responses. IMPORTANCE Ad-based vectors are widely used in experimental preclinical and clinical immunization studies against numerous infectious agents, such as human immunodeficiency virus, Ebola virus, Plasmodium falciparum , or Mycobacterium tuberculosis Preexisting immunity to Ad-based vectors is widely recognized as a hindrance to the widespread use of Ad-based vectors for immunizations in humans; however, our data show that an immune response to Ad-derived T cell epitopes can also result in loss or impairment of transgene-specific immune responses in prenaive vaccinees due to immune competition. Our results highlight that seemingly immunodominant epitopes may be affected by dominance of vector-derived epitopes, and modifications of the vector design or the immunogens employed in immunization may lead to more effective vaccines., (Copyright © 2017 American Society for Microbiology.)

While Friend retrovirus-infected mice readily mount a vigorous CD8(+) T cell response to the leader-gag-derived peptide GagL(85-93), no GagL(85-93)-specific T cells were detectable in mice immunized against Friend virus (FV) with viral vectors or DNA vaccines. By exchanging one epitope-flanking amino acid or using a scaffold protein we were able to demonstrate for the first time the induction of GagL(85-93)-specific CD8(+) T cells by genetic vaccination and show their high protective effect against FV challenge infection.