In the current report, we used different approaches to further improve the immunogenicity and protection efficacy of plague DNA vaccines expressing V or F1 alone, or F1 fused with YscF. The results showed that immunogenicity of bacterial antigens can be improved by using either a well-established approach, such as codon optimization or some other less studied approaches, such as antigen engineering.
The Y. pestis
genome harbors three plasmids necessary for full virulence of the bacterium besides the chromosome. Based on the analysis of genes with coding sequence equal to or larger than 300 bps, the overall “G+C” content was 47.64% in the whole Y. pestis
]. The synonymous codon usage pattern of these genes was different. Gene expression levels are strongly related to the frequency of “G+C” at the third position of synonymous codon usage [37
]. Sequence analysis for the Y. pestis
gene coding for V protein, lcrV, as part of the 70-kb low calcium response (lcr) plasmid, showed a “G+C” content of only 37.71%. Because codon usage is directly linked to preferred tRNA usage in a given cell type, it is conceivable that codon optimization for LcrV gene is important for its high level expression of this bacterial protein in mammalian cells [38
]. However, in a previous report, codon optimization for the V gene based on murine codon preference did not make any difference in a DNA vaccine vector pVAX1-CMV-TE when it was tested in mice and the peak anti-V antibody titers were measured [22
]. Results in the current study showed that codon optimization is very effective in not only eliciting higher antibody responses but also in improving protection when compared to the wild type V gene-based DNA vaccine.
There are several differences between these two studies. First, the exact levels of codon changes may be different, however, since no sequence information is available for the previous study, it is hard to compare the exact sequence difference between the two studies. In our study, we also made sure to minimize sequence changes that may generate inhibitory secondary RNA structures. It is not known whether a similar analysis was incorporated in the previous study. It is quite unusual that in the previous report that one group of mice immunized with the codon optimized V gene DNA vaccine had worse immune responses than the wild type V gene DNA vaccine. It appeared that some changes in the codon optimized V gene had a negative effect on the otherwise immunogenic wild type V gene. Second, the study approaches were different in these two reports. In the study conducted by Garmory et al., the comparison between the codon optimized and the wild type V DNA vaccines was done in mice using only intramuscular (im) immunization and mice required up to five im immunizations before any positive anti-V antibody titers could be detected [22
]. However, in our previous [25
] and current studies, strong positive anti-V IgG responses can be easily detected within two DNA immunizations delivered by a gene gun. It was shown recently that both the physical DNA delivery methods (gene gun or electroporation) were much more effective than the intramuscular needle injection method [39
]. Because the overall antibody levels were so low in the previous report [22
], a protein boost had to be given in order to detect any difference between codon optimized and wild type V genes when they were used as a prime immunization, which may bring more variables to the final measurement. Finally, in the current study, the protection results from the challenge study provide more definitive evidence to support the survival benefit of codon optimized V DNA vaccine while the previous study did not test such a difference in challenge studies between the vaccine formulations with different codon usages.
A different optimization approach was employed for the F1 antigen in the current study. F1 is a 17.5 kDa protein which is considered an important but not essential virulence factor unique to Y. pestis
. The reason for this is because the F1-negative mutant bacterial strains do not abolish virulence of Y. pestis
but lead to a delay in onset of the disease in animal models [40
]. Recombinant F1 protein is very immunogenic as are F1 DNA vaccines [24
]. Our previous study also demonstrated the addition of the leader sequence of human tissue plasminogen activator (tPA) in front of the entire original coding sequence for F1 was able to improve the immunogenicity of F1 DNA vaccine [25
], but a study from another group showed that the removal of a putative F1 leader sequence from the F1 coding sequence was more protective than adding a signal-bearing E3 polypeptide of Semliki Forest Virus [24
]. In order to reconcile such differences, we combined designs from the above two studies by using the leader of tPA in these studies.
The leader sequence of tPA has been shown in multiple DNA vaccine studies to produce more secreted downstream protein in mammalian expression systems. As a result, immunogenicity of many DNA vaccines with a tPA leader is also improved as shown by higher antibody responses including the envelope protein of HIV-1 [26
] and LcrV protein of Y. pestis
]. Because the original F1 gene has a short hydrophobic segment in its N-terminus, which may serve as a putative leader sequence for a bacterial protein, we tested the effect of adding a tPA leader to the F1 gene, with and without the removal of this putative F1 leader.
Adding a tPA leader to both types of F1 genes was able to increase the secretion of F1 protein, as shown by increased detection of F1 in culture supernatant, and to improve the levels of anti-V IgG responses. However, the protection results showed that the addition of a tPA leader led to lower protection when compared to counterparts without a tPA leader. This was the case between either the tPA-F1 and F1 pair or the tPA-dF1 and dF1 pair, i.e., removal or not of the putative F1 leader did not make the addition of a tPA leader more effective. This finding was further confirmed by the data that the protection levels for F1 (keeping the original putative leader) and tPA-dF1 (replacing the putative leader with a new tPA leader) were similar. The worst among the four F1 DNA vaccine designs was tPA-F1, which basically had two leaders (one from tPA plus the putative F1 leader). The most effective design was dF1, F1 without any N-terminal leader sequence. It is not clear why a “leader-less” F1 is more protective, given the finding that this design did not lead to the highest anti-F1 IgG responses. Future studies should analyze additional bacterial proteins to understand whether bacterial proteins have any unique sequences that may affect their expression in the mammalian system and, more importantly, their functional conformation in eliciting protective immune responses. This knowledge is important not only to DNA vaccines but also to recombinant protein-based bacterial vaccines, which only started to enter the vaccine development pipeline in recent years. DNA vaccine studies, as presented in the current report, can certainly be used as very valuable tools to any subunit-based vaccine studies.
Bacteria vaccines can take advantage of the large number of proteins associated with this type of pathogen by including multiple antigens in one vaccine formulation to improve the efficacy of a vaccine or to minimize the escape of vaccine-induced protection. However, technically, a polyvalent subunit-based vaccine can be challenging because of the cost and technical complexity associated with the production of multiple protective vaccine components. In the past, a V-F1 fusion protein was proposed to minimize such issues associated with physically separate, bivalent V and F1 protein vaccines. In the current report, we tested the same concept by making F1 and YscF fusion antigens, which were then tested by DNA immunization.
Our results indicate that DNA vaccines expressing F1 and YscF fusion proteins could induce antigen-specific antibody responses against both F1 and YscF antigens. Two versions of fusion antigens were produced, either F1 upstream of YscF or YscF upstream of F1, and both were immunogenic as evidenced by their ability to generate similar levels of antigen-specific antibody responses. However, improved protection was observed only when the YscF was fused downstream of F1 protein (F1-YscF design) while the other fusion antigen design (YscF-F1) had reduced protection when the tPA-F1 DNA vaccine was used as the baseline control for the protection studies using both fusion antigens. In our previous study, we demonstrated that YscF in its dimer form, but not in the monomer form, was protective [17
]. Therefore, we did not include a YscF monomer as the control in the current study. Even with the dimer form of YscF, the protection level was only partial (60% protection against intranasal challenge with 15 LD50 of Y. pestis
It is also interesting to observe that the YscF-F1 design had much higher levels of secreted antigens than the F1-YscF1, but could not induce better protective immunity than the latter. When the F1 antigen alone was included, we found that it was most protective when it had no leader sequence and did not require high level of secretion, as examined by in vitro
assays. Therefore, it may not be a total surprise that a mainly intracellularly expressed F1-YscF fusion protein is more protective than the easily secreted YscF-F1 fusion protein. However, this finding, along several other results presented in the current report, points to the importance of antigen engineering in the next phase of DNA vaccine research. In the past, given the gene-based nature of DNA vaccines, optimization of the DNA vaccine vector and delivery of DNA vaccines have been the main focus of research being conducted. The findings that changes to the coding sequence itself can significantly affect not only the immunogenicity but also protection will bring more attention to the designs of antigen inserts which may affect a wide range of considerations, including antigen conformation, antigen expression, post-translational antigen modification, and ultimately the fate of antigen processing and presentation in vivo
which remains largely unknown to DNA vaccines. Results reported here using Y. pestis
proteins as model antigens should make antigen engineering the first step of the above complicated process as we previously noted [42