Currently, there is no preventative treatment against the deadly hemorrhagic fever caused by EBOV infection. Due to the highly contagious and deadly nature of filoviruses, there is great concern that these lethal agents may be used as biological weapons or terrorism agents against civilized nations, in addition to the fear that these viruses may spread into populated urban areas as a result of increasing modern travel. Consequently, the development of effective EBOV vaccines to prevent the further evolution and spread of EBOV has become a great interest to many research groups.
In this study, we evaluate the first bivalent EBOV vaccine designed to prevent infection by both the Zaire and Sudan species. Our approach differs from other vaccine strategies, which are all monovalent in design and therefore express single antigens from only the Zaire species (3
). Among the many tested approaches in developing EBOV vaccines, vector-mediated antigen transfer, using either the first-generation adenoviral vector (21
) or a replication-competent vesicular stomatitis virus vector (6
), appears to be the most promising. Both studies demonstrated protection of nonhuman primates against ZEBOV challenges. However, one major difficulty that remains in the development of an effective EBOV vaccine is the requirement for a bivalent capability to induce protective immune responses against two EBOV species, Zaire and Sudan, which have been responsible for all human deaths due to EBOV infection thus far.
Sullivan et al. only partially addressed this issue by vaccinating guinea pigs with up to four plasmid vectors, each expressing a single-species EBOV GP, in a single injection (23
). While these animals survived challenge by ZEBOV, no data were shown to demonstrate that these animals developed either a humoral or a CMI response to any EBOV species other than Zaire. In addition, while the vesicular stomatitis virus-based vaccine was protective against ZEBOV challenge, it was unable to protect cynomolgus macaques from challenge with SEBOV (6
). Other current vaccine strategies such as Ebola virus-like particles have demonstrated protective efficacy (27
); however, these particles are inefficient to produce and would require cotransfection of several plasmids simultaneously to develop a bivalent vaccine approach. In addition, our vaccine demonstrated 100% protection of two mouse strains against viral challenge, while the alphavirus replicon (10
), baculovirus (8
), vaccinia virus (4
), and DNA plasmid (25
) approaches were all found to be only partially protective in small-animal models.
Importantly, there is a distinct difference between our cAdVax-based vaccines and those vaccines based on the first- generation Ad. The major advantage of the cAdVax system over the first-generation Ad vector is the ability to express multiple (up to six) antigens in a single construct. Upon vaccination, all of the antigens carried by the vector will be produced at high levels within the cells transduced at the site of vaccination. We hypothesize that vector-based vaccine gene transfer induces a de novo antigen synthesis, which results in a natural antigen expression and presentation on cell surfaces. This mimics a natural infection by the pathogenic viruses and induces potent immune responses without causing the disease.
Vaccines based on antigen synthesis de novo create a major advantage over protein-based subunit vaccines that are only capable of presenting linear epitopes. They also have an advantage over recombinant protein antigen synthesis in eukaryotic cells in which the correct conformation of the glycoproteins that contain the receptor-binding site may be destroyed in the extensive purification processes. In contrast, GP antigens synthesized de novo would theoretically retain the natural conformations and posttranslational modifications of the native GPs and therefore would include intact viral receptor-binding sites, where virus-neutralizing epitopes would be located.
Because we constructed cAdVax vaccines that expressed the GP of the Sudan [cAdVaxE(GPs)] or Zaire [cAdVaxE(GPz)] species of EBOV, as well as the bivalent cAdVaxE(GPs/z) vaccine that expresses the GP of both species of EBOV, we were able to study type-specific and cross-reactive immune responses and the true bivalent immunity against both species of virus. We have clearly shown that immune responses are specific to each species of EBOV, although some level of cross-reactivity between the two different species was observed in ELISA. The significance of the cross-immune responses detected by ELISA in broad immune protection is questionable. It is not known, in natural infections, whether patients who have recovered from one species of EBOV infection would develop protective immune responses against other species, due to the high mortality of the infection and the rareness of these diseases. However, in the nonhuman primate study conducted by Jones et al., monkeys challenged with ZEBOV were not protected against challenge by SEBOV (6
), thereby indicating the need for a bivalent vaccine designed to prevent infections by both species. Studies of cross protection have been further complicated by the lack of an effective neutralizing assay (5
). It is generally believed that neutralizing assays are not predictive of protection (5
), as protective immunity has been observed despite negative results from plaque reduction assays (32
In addition to antibody responses, the cell-mediated arm of the immune system is critically important in defense against virus infections. Activated T lymphocytes play an essential role in destroying infected cells, preventing viral replication, reducing viral load, and eventually eliminating the infection. In the case of filovirus infections, mortalities often occur before sufficient time is allowed for the activation of CMI responses. We hypothesize that activation of an EBOV-specific CMI response prior to exposure to EBOV would give the cellular arm of the immune system a chance to establish itself and proliferate quickly in the event of an infection.
In our study, we have shown that antigen synthesis de novo can effectively induce CMI responses against the EBOV GPs, based on ELISPOT analyses. We attribute this to the persistent stimulation of the immune system by exogenously induced EBOV antigen production and presentation. In combination with the humoral responses, we believe that this EBOV-specific CMI response will play important roles in protective immunity against EBOV.
Ultimately, induction of protective immune responses against EBOV infections is the main goal for any vaccine strategy. In this study, we were able to demonstrate 100% protection of two genetically distinct strains of mice (C57BL/6 and BALB/c) against a mouse-adapted ZEBOV challenge given at 30,000 times the LD50
. Currently, a mouse model for SEBOV has not yet been established, and a nonhuman primate model for SEBOV has also been unavailable until recently (6
). In future studies, we plan to include additional challenge tests with SEBOV in nonhuman primates to fully evaluate the bivalent capability of the vaccine. However, because our cAdVaxE(GPs/z) vaccine is capable of inducing immune responses against both SEBOV and ZEBOV that are similar to those the cAdVaxE(GPz) vaccine makes against ZEBOV, we believe that vaccination with cAdVaxE(GPs/z) has the potential to protect animal models from EBOV infections by both species. Further tests will be necessary to determine whether this holds to be true.
To our knowledge, this is the first demonstration of a bivalent EBOV vaccine to coexpress multiple serotype proteins in a single vaccine construct, eliciting efficient humoral and cellular immune responses to both SEBOV and ZEBOV antigens. Among the many advantages of the cAdVax vaccine platform is its ability to express multiple antigens in a single vaccine construct, thereby simplifying the production and approval processes that would be necessary to bring a final Ebola virus vaccine to the public. In establishing a vaccine comprised of a single vaccine vector, this decreases production costs and FDA approval costs as well as ensuring that each transduced cell expresses all incorporated antigens at a 1:1 ratio. Importantly, the cAdVax vaccine demonstrated efficient induction of a protective immune response, demonstrating 100% protection of two strains of mice against lethal EBOV challenge. Taken together, our data suggest that a cAdVax-based multiple antigen vaccine, such as cAdVaxE(GPs/z), represents a promising candidate for the development of an effective bivalent vaccine against EBOV infections.