DNA-Based Vaccine
Live attenuated viruses and recombinant proteins have been used successfully in a variety of vaccines, but the safety and immunogenicity of gene-based vaccines have proven increasingly attractive. Among the gene-based approaches, naked plasmid DNA has been used successfully in animal models to direct the synthesis of immunogens within the host cells and has proven helpful in a variety of infectious diseases, for example, influenza, malaria, tuberculosis and Hepatitis B. Previous attempts to elicit protective immune responses against Ebola virus by using traditional active and passive immunization approaches have not succeeded. Elicitation of anti-EBOV viral protein antibodies appears to be a desirable objective for a vaccine, but it may not be sufficient for protection and may not even be the primary means of protection.
EBOV glycoprotein (GP) is the only protein known to be on the Ebola virion surface. If a strong cell-mediated immune response is also needed for effective protection from EBOV infections, then other proteins such as nucleoprotein (NP) may also prove useful.
Genetic immunization with plasmid DNA was developed in the guinea pig and was the first successful vaccine for Ebola virus.
The DNA plasmids containing GP, soluble glycoprotein (sGP) and NP were first injected into mice to characterize the ability to induce humoral and/or cellular responses to the relevant viral proteins. Three injections were performed at 2-week intervals in BALB/c female 6-8 week old mice. In this model, NP induced a readily-detectable high antibody titer. Despite the substantial humoral immune response to NP, minimal CTL activity was detected against syngeneic cells expressing this viral protein. In contrast, genetic immunization with sGP, which elicited a slightly weaker antibody titer, induced a marked cytolytic T-cell response to cells expressing GP. Immunization with the GP plasmid also induced a significant CTL response to GP. These data suggested that both the secreted and transmembrane forms of the protein could be processed for antigen presentation and that the transmembrane form was a target for recognition by these CTLs. Also, antigen-specific T-cell proliferation to sGP was also observed in mice injected with GP and sGP but not with control plasmid.
To determine whether the in vivo immune responses could protect against viral infection, the virus was adapted to grow in guinea pigs. In this model, the animals were injected intramuscularly into each hind leg with the relevant expression vector plasmids or control plasmids. Two groups of immunized guinea pigs were studied. In the first group, animals challenged within 2 months after the initial immunization had high antibody titers and nearly complete protection from lethal challenge was observed in GP, sGP and NP immunized subjects, in contrast to controls. In the second group, guinea pigs were challenged 4 months after the initial immunization. Antibody titers for all three viral protein DNA vaccines were lower. In this group, guinea pigs that expressed GP or sGP was conferred much more effective protection than NP.
These results suggest that a T-cell proliferative immune response and marked CTL activity correlates with increased survival in guinea pigs, conferring longer-lasting and hence more effective protection against lethal challenge of Ebola virus.
NP and GP DNA vaccines conferred protective immunity in mice only after multiple doses of the vaccine.
To determine if the GP DNA was able to elicit protective immunity in mice, BALB/c mice were vaccinated by delivering DNA-coated gold beads to the abdominal epidermis by particle bombardment with helium pressure using a gene gun. Mice that were primed and then boosted multiple times two months before challenge survived the viral challenge. Although GP alone offered protection from EBOV challenge, at least four vaccinations were required. For a human vaccine, it is desirable to elicit immunity with the lowest possible number of vaccinations. This mode of vaccination is not feasible in humans because giving multiple injections not only increases the cost of the vaccine but also runs the risk of tolerizing the immune system to the virus.
To compare the vaccine potential of NP and GP, mice were inoculated with each of the two candidate vaccines. Antibodies were detectable after the first inoculation with the first vaccine, increasing with each of the two subsequent inoculations. Mice were challenged one month after the final vaccination and interestingly, only partial protection was achieved with both GP and NP vaccines, although CTL activity and high antibody titers were detected.
A DNA priming-boosting strategy using replication-defective adenovirus for an Ebola virus vaccine was highly successful in cynomolgus macaques.
While DNA vaccines have been highly effective in the guinea pig model, their efficacy in nonhuman primates or humans has been less impressive. Priming-boosting immunization protocols that use DNA immunization followed by boosting with poxvirus vectors carrying the genes for pathogen proteins have yielded dramatically enhanced immune responses in animal studies, with 30-fold or greater increases in antibody titer from the booster, as been reported for a malaria DNA vaccination with a booster of modified vaccinia virus. This experiment showed that DNA priming followed by MVA boosting may provide a general immunization regime for induction of high levels of CD8+ T cells. More importantly, this approach had been used to achieve successful protection against the Marburg virus, a member of the filovirus family as well, using a GP-based alphavirus-replicon vaccine. At that point, adenoviruses (ADV) had been shown to have a boosting effect in mice, but the combination of DNA and adenovirus had not been tested for efficacy in an infectious challenge model, and the success of this approach in primates was not known, hence in 2000, Sullivan et al proceeded to determine this for EBOV-challenged Cynomolgus macaques.
In this model, Cynomolgus macaques were immunized intramuscularly twice with DNA encoding the GPs of ZEBOV, SEBOV and ICEBOV, and NP of ZEBOV first. This was followed by a booster of adenovirus expressing ZEBOV GP five months after priming. The monkeys were challenged with six plaque-forming units (PFU) of ZEBOV intramuscularly and all four animals survived after challenge. Antibody responses, T-cell proliferation and CTL responses indicated that antibody and T memory helper cells are essential for the protection, and that cell-mediated immunity may be important. This study demonstrated the superior immunologic efficacy of this priming-boosting combination for both cellular and humoral responses. These animals displayed complete immune protection against a lethal challenge of virus, providing the first demonstration of an Ebola virus vaccine approach that protects primates against infection.
Accelerated vaccination for Ebola virus has been developed in Cynomolgus macaques8.
Although the DNA-priming/boosting vaccine has been shown to successfully protect Cynomolgus macaques against Ebola infection, more than six months was required to complete the immunizations, making it impractical to limit an acute epidemic. Thus, an accelerated vaccine was sought.
In the experiments carried out by Sullivan et al in 2003, Cynomolgus macaques were immunized with ADV-GP and ADV-NP, followed by boosting 9 weeks later. One week after the boost, animals were challenged with either low or high PFU of ZEBOV. These doses were uniformly fatal 6-12 days afterwards in saline-injected control animals. In contrast, the ADV-GP/NP immunized monkeys were completely protected, confirmed by viral load. The antibody response to immunization with ADV-GP/NP was induced more rapidly than with DNA priming and ADV boosting, but it was of a lower magnitude. Protection was highly effective and correlated with the generation of Ebola-specific CD8+ T-cell and antibody responses. Even when animals were immunized once with ADV-GP/NP and challenged 28 days later, they remained resistant to challenge with either low or high dose of virus.
This accelerated vaccine thus provides an intervention that confers protection after a single immunization8. If this vaccine works similarly in humans, it may be useful in the containment of acute outbreaks by ring vaccination.
Propositions for a better DNA vaccine :
1. ADV-GP/NP + adjuvant of CpG DNA
CpG DNA has been shown to be important in its ability to drive strong Th1-like
T-cell responses and is an extremely potent stimulus to dendritic cells, inducing them
to make high levels Th1-like cytokines IL-12 and IL-18, to express co-stimulatory
molecules and to have increased ability to activate T cells. Thus, CpG DNA can be
used as an effective adjuvant for inducing strong Th1-like responses with CTLs,
inducing potentially greater protective immunity against EBOV infection.
2. ADV-GP+NP
Instead of adding GP or NP DNA into the ADV vector, both GP and NP could be simultaneously included in the vector construct. Immune response from each viral proteins could act synergistically to elicit an even larger and more rapid antibody, T-cell proliferation and EBOV-specific CTL response.
3. ADV-putative conserved motif
Sequencing all known Ebola virus strains and screening for conserved motifs throughout the strains and placing these conserved motifs in an adenovirus vector could possibly render an individual immune to all of the Ebola strains with one immunization.
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This page last updated: 14 April 2004.
