**4. Comparison to Conventional DNA Immunization**

In attempts to evaluate the feasibility of alphavirus DNA replicons as vaccine vectors, a direct comparison to conventional DNA vaccines has been an essential component. In this context, both the conventional DNA plasmid pWRG7077 and the SIN DNA replicon expressing SEOV M and S gene segments showed potential as vaccine vectors as described above [35]. However, there were substantial and to some extent surprising differences. In vitro expression levels were consistently higher from the conventional DNA vector than from the SIN DNA replicon. However, higher titers were obtained in vivo for vaccinations with SIN DNA replicons than for the conventional DNA plasmid. It has been suggested that the enhanced immune response relates to certain alphavirus vector genes promoting cell death and inducing interferon responses [41]. Moreover, as described above, immunization with SIN-TRP1 DNA broke tolerance and provided immunity to melanoma, which was not the case for conventional DNA vaccines [30]. Similarly, the long-term protection against *M. tuberculosis* obtained by immunization with SIN-Ag85 DNA was not achieved by vaccination with a conventional DNA plasmid in mice [21].

Several studies have demonstrated that in general, significantly lower doses of alphavirus DNA replicon are required to achieve the same level of response as seen for conventional DNA vaccines [14,17]. For instance, 100-fold to 1000-fold lower doses of SIN-HSV-1-gB were needed to elicit antibody responses and protection against lethal virus challenges. Moreover, a single dose of 10 ng elicited strong immune responses in mice. In the context of cervical cancer vaccines, while a conventional DNA-based vaccine failed to prevent tumor growth, immunization with a 200-fold lower equimolar dose of 0.05 μg of the SFV DNA replicon resulted in complete tumor regression in 85% of immunized mice [26]. In attempts to enhance the immune responses, the alphavirus DNA replicon vector expressing the multiclade HIV-1 T cell immunogen HIVconsv (DREP.HIVconsv) was subjected to intradermal delivery followed by in vivo electroporation and compared to the conventional DNA plasmid pTH.HIVconsv [42]. HIV-1-specific CD8<sup>+</sup> T cell responses were obtained in mice with 1 μg of pTH.HIVconsv compared to only 3.2 ng of DREP.HIVconsv, which represents a 625-fold molar dose reduction. These responses could be further enhanced for both the conventional DNA plasmid and the alphavirus DNA replicon by heterologous vaccine boosts with MVA-HIVconsv and attenuated chimpanzee adenovirus ChAdV63.HIVconsv. Additionally, immunization of rhesus macaques demonstrated that application of alphavirus DNA replicon vectors allowed to reduce the dose by at least 20-fold compared to conventional plasmid DNA vectors. For this reason, the manufacturing of large batches of GMP grade material for clinical trials and marketed products is easier and more feasible. Another feature of importance related to DNA replicon vaccines is that the expression is transient and lytic, eliminating such biosafety risks as chromosomal integration and the induction of immunological tolerance [43].

#### **5. Conclusions**

Several studies have confirmed that alphavirus DNA replicon vectors elicit strong immune responses in vaccinated animal models targeting both infectious agents and tumor antigens. Moreover, protection against lethal challenges by viruses, bacteria, and tumor cells have also been established. In many cases, DNA replicon vaccines have proven superior to conventional DNA plasmid vaccines or at least as efficient. However, it has been confirmed that significantly lower doses of DNA replicon vaccines are needed to achieve the same immune responses and protection as for conventional DNA vaccines. In the context of alphaviruses, in addition to DNA replicon vectors, RNA replicons and recombinant alphavirus particles have also been subjected to vaccine studies. So far, there is no clear indication of which delivery format is the best and it seems more like the ranking order varies from one target to another.

Related to the biosafety of DNA vaccines, the probability of stable chromosomal integration of transfected DNA presents some concern. In this context, it was confirmed that an intramuscularly administered DNA vector expressing a luciferase reporter gene could be detected in the skeletal muscle for more than 19 months [44]. However, the DNA was only present as an extrachromosomal plasmid. When intramuscular immunization was followed by electroporation, low-level random chromosomal integration occurred, although the frequency was significantly lower than observed for spontaneous gene mutations [45]. Another study demonstrated that DNA administration into the skeletal muscles resulted in the presence of a majority of the DNA at the injection site with only minor amounts detected in other organs [46]. Moreover, no genomic plasmid DNA integration was discovered. Related to immune responses, no anti-DNA antibodies were observed after repeated intramuscular injections in primates [47]. Another issue relates to the presence of prokaryotic elements such as antibiotic resistance genes in DNA vaccines [48]. However, no transfer of such elements has been documented so far.

Another concern of alphavirus DNA replicon vaccines relates to the difficulties in transferring the strong immune responses detected in rodents to larger animals and most importantly to humans. Disappointingly, this has also been verified in clinical trials which have supported the need of

dose optimization [49–51]. Recent studies have indicated that prime-boost strategies combining alphavirus-based vaccines with other viral-based vaccines have enhanced the immunogenicity, which is important, especially in clinical settings. Another approach briefly mentioned in this review relates to the improved delivery and stability of DNA-based vaccines through polymer and lipid encapsulation procedures. Moreover, efforts are being made to target dendritic cells in order to generate better immune responses for future vaccines. Overall, alphavirus-based DNA vaccines have the potential to provide a flexible and inexpensive alternative to current existing approaches.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The author declares no conflict of interest.


*2.1. Patient Information*

All procedures and protocols for this study were reviewed and approved by the Colorado Multi-Institutional Review Board (COMIRB 17-1136). Subjects included in this study were patients undergoing repeat resective surgery after radiologically defined tumor progression between August and November 2018 at the University of Colorado Hospital. The patient set consisted of 10 subjects who received prior resection(s) for recurrent glioma with detailed histopathology recorded for 2 or more biopsies (41 biopsies collected in total). Data were collected retrospectively from patient chart review. Two patients received two prior resections; all others received one prior resection. The patient set is divided into two groups: those that underwent radiation therapy prior to repeat resection (RT, *n* = 7) and those that did not (No RT, *n* = 3). For each patient, biopsy samples were collected during surgery from the radiologically-defined tumor region and examined by an expert neuropathologist (B.K.D.). The neuropathologist classified each sample and an expert neurosurgeon (D.R.O.) designated each classification as primarily consisting of abnormal, nontumor tissue (Abnormal), or tumor tissue (Tumor). Patient information is summarized in Table 1.

Abbreviations: M = male, F = female, MT = mutant, WT = wild type, NA = not available, lo = low expression, hi = high expression, + = methylated, − = unmethylated, IDH = isocitrate dehydrogenase, MGMT = O-6-methylguanine-DNA-methyltransferase, EGFR = epidermal growth factor receptor, BRAF = v-Raf murine sarcoma viral oncogene homolog B, RT = radiation therapy, CT = chemotherapy.

#### *2.2. Imaging Sequence Parameters*

All images were obtained using a 3.0-T whole-body MR imager (Signa HDxt; GE Medical Systems, Milwaukee, Wisconsin, USA) between 0–24 days prior to repeat surgical intervention. Acquisition times were 2.5, 5.4, 4.6, 7.8, and 9.0 minutes for non-enhanced T1-weighted (T1w), gadolinium-enhanced T1-weighted (T1ce), T2-weighted (T2w), T2-FLAIR (FLAIR), and diffusion-weighted (DW) images, respectively. For T1w, TE = 2.3 ms, TR = 5.5 ms, and flip angle = 8◦. Data were recorded as a 256 × 256 matrix with 1 mm × 1 mm pixel spacing, a slice thickness of 1.2 mm, and zero slice gap. For T1ce, TE = 2.5 ms, TR = 6.8 ms, and flip angle = 8◦. Data were recorded as a 512 × 512 matrix with 0.5 mm × 0.5 mm pixel spacing, a slice thickness of 1.2 mm, and zero slice gap. For T2w, TE = 6333 ms,

TR = 80 ms, and flip angle = 142◦. Data were recorded as a 512 × 512 matrix with 0.5 mm × 0.5 mm pixel spacing, a slice thickness of 2 mm, and zero slice gap. For FLAIR, TE = 6000 ms, TR = 128 ms, and flip angle = 90◦. Data were recorded as a 512 × 512 matrix with 0.5 mm × 0.5 mm pixel spacing, a slice thickness of 1.2 mm, and zero slice gap. For DW images, TE = 85 ms, TR = 16,000 ms, and flip angle = 90◦. The diffusion gradient was encoded in 32 directions at b = 1000 s/mm2 and an additional measurement without the diffusion gradient (b = 0 s/mm2). DW data were recorded as a 128 <sup>×</sup> 128 matrix with 0.9375 mm × 0.9375 mm pixel spacing. A total of 50 sections were obtained with a slice thickness of 2.6 mm and zero slice gap.