Treatment of HCV infection has recently benefited from the addition of protease inhibitors used in combination with PEG–IFN-α/ribavirin, leading to improvement in cure rate reaching up to 70%–80% for genotype 1–infected patients in clinical trials. However, problems such as increased cost, increased side effects, and difficulties to treat specific populations still remain. Therapeutic vaccines represent a novel class of compounds displaying a mechanism of action complementary to those of direct acting antivirals [32
]. The most advanced HCV therapeutic vaccines to date are in phase I/IIb clinical development, and, although it is expected that their primary positioning will be in combination with antivirals, it remains interesting to develop an improved vaccine regimen that may cure patients without addition of drugs. The threshold of immunity required to reach such result will likely have to be greatly enhanced. Prime/boost strategies based on DNA vaccines and viral vectors have emerged as a powerful approach in various infectious models to improve the number of antigen-specific T cells and to increase efficacy in challenge assays [34
]. In the HCV field, prime/boost approaches have been developed with some success in animal models using DNA, adenovirus, or polypeptides as primer and canarypox viruses, DNA, adenovirus or alphaviral particles as booster vaccines [36
]. However, the only prime/boost study so far to reach the clinic in the HCV field is the human adenovirus 6 (Ad6) prime and chimpanzee adenovirus 3 (Ad3) boost approach that activated T cells specific for multiple HCV proteins with secretion of Th1-type cytokines [39
In this proof-of-concept study, we compared established vaccine schedules shown earlier to result in potent CD8+
T-cell immunity both for MVATG16643 (3 injections 1 week apart) and for ChronVac-C (2 injections 4 weeks apart) [15
], with the combined use of these 2 vaccines in prime/boost settings. The DNA vaccine was first administered, followed by boosting with the MVA vaccine (with a 5- or a 12-week time interval). To provide an exhaustive analysis, we used both C57BL/6J mice and “humanized” HLA-A2 transgenic mice. Immune responses induced were analyzed by the use of CD8+
-restricted epitopes, peptide libraries, and proteins derived from all 3 NS3, NS4A/B, and NS5B antigens from both genotype 1a and genotype 1b vaccine sequences.
A superiority of the ChronVac-C prime/MVATG16643 boost over either of the vaccine used alone was suggested at different levels. First, pentamer staining of GAV-specific responses in C57BL/6J demonstrated a considerable expansion of CD8+ T cells up to 23% following the prime/boost vs 7.7% at best for single vaccine–based strategies. This improvement did translate to enhancement at the functional level. ELISpot assay showed, for example, that the heterologous prime/boost induces in C57BL/6J mice a GAV-specific response 3.5-fold higher than that induced by ChronVac-C or MVATG16643 alone. In HLA-A2 mice, IFN-γ–producing CD4+ T cells specific for FPNS34A 1a-2 epitope were 6.5- to 8.7-fold higher than responses developed after ChronVac-C or MVATG16643 immunization. This is a key feature in the development of an immune-based therapy against chronic HCV infection where both CD4+ and CD8+ arms of the immune response have been found to be altered.
Numerous studies have shown that the quality even more than the level of T-cell responses is crucial for determining the outcome of various infections [40
]. For HCV, it was even reported that resolution obtained under early interferon therapy correlated with the development of polyfunctional IFN-γ/IL-2/CD107α+
T cells [42
]. Whereas ChronVac-C or MVATG16643 alone and homologous MVATG16643 prime/boost were able to induce 2%–3.5% of trifunctional IFN-γ/TNF-α/CD107α+
T cells, ChronVac-C prime/MVATG16643 boost resulted in the striking detection of this population up to 29% of CD8+
T-cells. In addition, the heterologous prime/boost induced up to 7.2% of quadrifunctional T cells (producing also IL-2). These results reenforced the superiority of the heterologous prime/boost regimen not only in terms of magnitude of induced T-cell responses but also in terms of their polyfunctionality.
The spectrum of epitopes recognized was also improved by the ChronVac-C/MVATG16643 vaccination in HLA-A2 mice. This was due first to ChronVac-C–specific induction of anti–CVN/CIN responses or MVATG16643-specific induction of anti-NS5B responses; thus expected additive effects were observed. More remarkable was that potential synergy was observed for the CD4+
-driven responses beyond the simple addition of values expected with each vaccine alone (see IFN-γ ELISpot and IFN-γ/TNF-α ICS data with E13K, NS3, and FPNS34A 1b and 1a stimulations). Several features of the DNA and the MVA vectors may underlie their synergic activities. DNA vaccines introduce encoded proteins into MHC class I and II antigen-processing pathways [18
], whereas its nonreplicative nature focuses immune responses on the encoded antigens [34
]. In contrast, MVAs express higher levels of encoded antigens, and T-cell responses induced seem to be dominated by cross-priming in vivo, despite the ability of the virus to infect antigen-presenting cells [43
Finally, beyond extensive quantitative and qualitative characterization, it was also important to address the in vivo functionality of the generated immune responses. For this purpose we utilized the Listeria
surrogate challenge model, which allow us to determine migration of vaccine-primed T cells to the Listeria
-infected organs, thereby monitoring eradication of antigen-expressing cells [15
]. We showed that the heterologous prime/boost regimen conferred significant protection against a challenge with a Listeria-
expressing HCV-NS3 protein, whereas none of the single vaccine–based regimens succeeded in a significant control of the bacteria replication. Notable is the variation in bacterial load within groups of mice, although it is in a similar range as seen in previous studies [15
]. Collectively, all immunological data in this study favor a correlation between the strength of the induced T-cell response and protection against Listeria
In conclusion, this study demonstrated the clear interest of the ChronVac-C prime/MVATG16643 boost strategy in agreement with reports arguing in favor of heterologous vaccine approaches. But results also go beyond: we used for the first time in the HCV field 2 vaccines already in clinical development. These vaccines express heterologous sequences derived from the 2 viral subtypes 1a and 1b belonging to the worldwide most prominent and hardest to treat genotype 1. We showed that the number of IFN-γ– and IL-2–producing CD4+ and CD8+ T cells, the epitope polyspecificity, and the polyfunctionality of induced CD8+ T cells were all greatly improved. In addition, vaccine-educated responses displayed protective function in vivo. These preclinical data support further evaluation of this vaccine combination in the clinic.