Pseudocowpox virus, a novel vector to enhance the therapeutic efficacy of antitumor vaccination.

Objective: Antitumor viral vaccines, and more particularly poxviral vaccines, represent an active field for clinical development and translational research. To improve the efficacy and treatment outcome, new viral vectors are sought, with emphasis on their abilities to stimulate innate immunity, to display tumor antigens and to induce a specific T-cell response. Methods: We screened for a new poxviral backbone with improved innate and adaptive immune stimulation using IFN-α secretion levels in infected PBMC cultures as selection criteria. Assessment of virus effectiveness was made in vitro and in vivo. Results: The bovine pseudocowpox virus (PCPV) stood out among several poxviruses for its ability to induce significant secretion of IFN-α. PCPV produced efficient activation of human monocytes and dendritic cells, degranulation of NK cells and reversed MDSC-induced T-cell suppression, without being offensive to activated T cells. A PCPV-based vaccine, encoding the HPV16 E7 protein (PCPV-E7), stimulated strong antigen-specific T-cell responses in TC1 tumor-bearing mice. Complete regression of tumors was obtained in a CD8+ T-cell-dependent manner after intratumoral injection of PCPV-E7, followed by intravenous injection of the cancer vaccine MVA-E7. PCPV also proved active when injected repeatedly intratumorally in MC38 tumor-bearing mice, generating tumor-specific T-cell responses without encoding a specific MC38 antigen. From a translational perspective, we demonstrated that PCPV-E7 effectively stimulated IFN-γ production by T cells from tumor-draining lymph nodes of HPV+-infected cancer patients. Conclusion: We propose PCPV as a viral vector suitable for vaccination in the field of personalised cancer vaccines, in particular for heterologous prime-boost regimens.

Evaluation of heterologous prime-boost vaccination strategies using chimpanzee adenovirus and modified vaccinia virus for TB subunit vaccination in rhesus macaques.

Tuberculosis (TB) still is the principal cause of death from infectious disease and improved vaccination strategies are required to reduce the disease burden and break TB transmission. Here, we investigated different routes of administration of vectored subunit vaccines based on chimpanzee-derived adenovirus serotype-3 (ChAd3) for homologous prime-boosting and modified vaccinia virus Ankara (MVA) for heterologous boosting with both vaccine vectors expressing the same antigens from Mycobacterium tuberculosis (Ag85B, ESAT6, Rv2626, Rv1733, RpfD). Prime-boost strategies were evaluated for immunogenicity and protective efficacy in highly susceptible rhesus macaques. A fully parenteral administration regimen was compared to exclusive respiratory mucosal administration, while parenteral ChAd3-5Ag prime-boosting and mucosal MVA-5Ag boosting were applied as a push-and-pull strategy from the periphery to the lung. Immune analyses corroborated compartmentalized responses induced by parenteral versus mucosal vaccination. Despite eliciting TB-specific immune responses, none of the investigational regimes conferred a protective effect by standard readouts of TB compared to non-vaccinated controls, while lack of protection by BCG underpinned the stringency of this non-human primate test modality. Yet, TB manifestation after full parenteral vaccination was significantly less compared to exclusive mucosal vaccination.

By Binding CD80 and CD86, the Vaccinia Virus M2 Protein Blocks Their Interactions with both CD28 and CTLA4 and Potentiates CD80 Binding to PD-L1.

In this article we report that the M2 protein encoded by the vaccinia virus is secreted as a homo-oligomer by infected cells and binds two central costimulation molecules, CD80 (B7-1) and CD86 (B7-2). These interactions block the ligation of the two B7 proteins to both soluble CD28 and soluble cytotoxic T-lymphocyte associated protein 4 (CTLA4) but favor the binding of soluble PD-L1 to soluble CD80. M2L gene orthologues are found in several other poxviruses, and the B7-CD28/CTLA4 blocking activity has been identified for several culture supernatants of orthopoxvirus-infected cells and for a recombinant myxoma virus M2 protein homolog (i.e., Gp120-like protein, or Gp120LP). Overall, these data indicate that the M2 poxvirus family of proteins may be involved in immunosuppressive activities broader than the NF-κB inhibition already reported (R. Gedey, X. L. Jin, O. Hinthong, and J. L. Shisler, J Virol 80:8676-8685, 2006, https://doi.org/10.1128/JVI.00935-06). A Copenhagen vaccinia virus with a deletion of the nonessential M2L locus was generated and compared with its parental virus. This M2L-deleted vaccinia virus, unlike the parental virus, does not generate interference with the B7-CD28/CTLA4/PD-L1 interactions. Moreover, this deletion did not affect any key features of the virus (in vitro replication, oncolytic activities in vitro and in vivo, and intratumoral expression of a transgene in an immunocompetent murine model). Altogether, these first results suggest that the M2 protein has the potential to be used as a new immunosuppressive biotherapeutic and that the M2L-deleted vaccinia virus represents an attractive new oncolytic platform with an improved immunological profile.IMPORTANCE The vaccinia virus harbors in its genome several genes dedicated to the inhibition of the host immune response. Among them, M2L was reported to inhibit the intracellular NF-κB pathway. We report here several new putative immunosuppressive activities of M2 protein. M2 protein is secreted and binds cornerstone costimulatory molecules (CD80/CD86). M2 binding to CD80/CD86 blocks their interaction with soluble CD28/CTLA4 but also favors the soluble PD-L1-CD80 association. These findings open the way for new investigations deciphering the immune system effects of soluble M2 protein. Moreover, a vaccinia virus with a deletion of its M2L has been generated and characterized as a new oncolytic platform. The replication and oncolytic activities of the M2L-deleted vaccinia virus are indistinguishable from those of the parental virus. More investigations are needed to characterize in detail the immune response triggered against both the tumor and the virus by this M2-defective vaccinia virus.

A multi-antigenic MVA vaccine increases efficacy of combination chemotherapy against Mycobacterium tuberculosis.

Despite the existence of the prophylactic Bacille Calmette-Guérin (BCG) vaccine, infection by Mycobacterium tuberculosis (Mtb) remains a major public health issue causing up to 1.8 million annual deaths worldwide. Increasing prevalence of Mtb strains resistant to antibiotics represents an urgent threat for global health that has prompted a search for alternative treatment regimens not subject to development of resistance. Immunotherapy constitutes a promising approach to improving current antibiotic treatments through engagement of the host's immune system. We designed a multi-antigenic and multiphasic vaccine, based on the Modified Vaccinia Ankara (MVA) virus, denoted MVATG18598, which expresses ten antigens classically described as representative of each of different phases of Mtb infection. In vitro analysis coupled with multiple-passage evaluation demonstrated that this vaccine is genetically stable, i.e. fit for manufacturing. Using different mouse strains, we show that MVATG18598 vaccination results in both Th1-associated T-cell responses and cytolytic activity, targeting all 10 vaccine-expressed Mtb antigens. In chronic post-exposure mouse models, MVATG18598 vaccination in combination with an antibiotic regimen decreases the bacterial burden in the lungs of infected mice, compared with chemotherapy alone, and is associated with long-lasting antigen-specific Th1-type T cell and antibody responses. In one model, co-treatment with MVATG18598 prevented relapse of the disease after treatment completion, an important clinical goal. Overall, results demonstrate the capacity of the therapeutic MVATG18598 vaccine to improve efficacy of chemotherapy against TB. These data support further development of this novel immunotherapeutic in the treatment of Mtb infections.

A Novel MVA-Based Multiphasic Vaccine for Prevention or Treatment of Tuberculosis Induces Broad and Multifunctional Cell-Mediated Immunity in Mice and Primates.

Bacille Calmette-Guérin (BCG) vaccination of new born babies can protect children against tuberculosis (TB), but fails to protect adults consistently against pulmonary TB underlying the urgent need to develop novel TB vaccines. Majority of first generation TB vaccine candidates have relied on a very limited number of antigens typically belonging to the active phase of infection. We have designed a multi-antigenic and multiphasic vaccine, based on the Modified Vaccinia Ankara virus (MVA). Up to fourteen antigens representative of the three phases of TB infection (active, latent and resuscitation) were inserted into MVA. Using three different strains of mouse (BALB/c, C57BL/6 and C3H/HeN), we show that a single vaccination results in induction of both CD4 and CD8 T cells, displaying capacity to produce multiple cytokines together with cytolytic activity targeting a large array of epitopes. As expected, dominance of responses was linked to the mouse haplotype although for a given haplotype, responses specific of at least one antigen per phase could always be detected. Vaccination of non-human primates with the 14 antigens MVA-TB candidate resulted in broad and potent cellular-based immunogenicity. The remarkable plasticity of MVA opens the road to development of a novel class of highly complex recombinant TB vaccines to be evaluated in both prophylactic and therapeutic settings.

Immunological characterization of a modified vaccinia virus Ankara vector expressing the human papillomavirus 16 E1 protein.

Women showing normal cytology but diagnosed with a persistent high-risk human papillomavirus (HR-HPV) infection have a higher risk of developing high-grade cervical intraepithelial neoplasia and cervical cancer than noninfected women. As no therapeutic management other than surveillance is offered to these women, there is a major challenge to develop novel targeted therapies dedicated to the treatment of these patients. As such, E1 and E2 antigens, expressed early in the HPV life cycle, represent very interesting candidates. Both proteins are necessary for maintaining coordinated viral replication and gene synthesis during the differentiation process of the epithelium and are essential for the virus to complete its normal and propagative replication cycle. In the present study, we evaluated a new active targeted immunotherapeutic, a modified vaccinia virus Ankara (MVA) vector containing the E1 sequence of HPV16, aimed at inducing cellular immune responses with the potential to help and clear persistent HPV16-related infection. We carried out an extensive comparative time course analysis of the cellular immune responses induced by different schedules of immunization in C57BL/6 mice. We showed that multiple injections of MVA-E1 allowed sustained HPV16 E1-specific cellular immune responses in vaccinated mice and had no impact on the exhaustion phenotype of the generated HPV16 E1-specific CD8⁺ T cells, but they led to the differentiation of multifunctional effector T cells with high cytotoxic capacity. This study provides proof of concept that an MVA expressing HPV16 E1 can induce robust and long-lasting E1-specific responses and warrants further development of this candidate.