Immune Requirements of Post-Exposure Immunization with Modified Vaccinia Ankara of Lethally Infected Mice

Current prophylactic vaccines work via the induction of B and T cell mediated memory that effectively control further replication of the pathogen after entry. In the case of therapeutic or post-exposure vaccinations the situation is far more complex, because the pathogen has time to establish itself in the host, start producing immune-inhibitory molecules and spread into distant organs. So far it is unclear which immune parameters have to be activated in order to thwart an existing lethal infection. Using the mousepox model, we investigated the immunological mechanisms responsible for a successful post-exposure immunization with modified vaccinia Ankara (MVA). In contrast to intranasal application of MVA, we found that intravenous immunization fully protected mice infected with ectromelia virus (ECTV) when applied three days after infection. Intravenous MVA immunization induced strong innate and adaptive immune responses in lethally infected mice. By using various gene-targeted and transgenic mouse strains we show that NK cells, CD4 T cells, CD8 T cells and antibodies are essential for the clearance of ECTV after post-exposure immunization. Post-exposure immunization with MVA is an effective measure in a murine model of human smallpox. MVA activates innate and adaptive immune parameters and only a combination thereof is able to purge ECTV from its host. These data not only provide a basis for therapeutic vaccinations in the case of the deliberate release of pathogenic poxviruses but possibly also for the treatment of chronic infections and cancer

Immediate-Early Expression of a Recombinant Antigen by Modified Vaccinia Virus Ankara Breaks the Immunodominance of Strong Vector-Specific B8R Antigen in Acute and Memory CD8 T-Cell Responses ▿

Efficient T-cell responses against recombinant antigens expressed by vaccinia virus vectors require expression of these antigens in the early phase of the virus replication cycle. The kinetics of recombinant gene expression in poxviruses are largely determined by the promoter chosen. We used the highly attenuated modified vaccinia virus Ankara (MVA) to determine the role of promoters in the induction of CD8 T-cell responses. We constructed MVA recombinants expressing either enhanced green fluorescent protein (EGFP) or chicken ovalbumin (OVA), each under the control of a hybrid early-late promoter (pHyb) containing five copies of a strong early element or the well-known early-late p7.5 or pS promoter for comparison. In primary or cultured cells, EGFP expression under the control of pHyb was detected within 30 min, as an immediate-early protein, and remained higher over the first 6 h of infection than p7.5- or pS-driven EGFP expression. Repeated immunizations of mice with recombinant MVA expressing OVA under the control of the pHyb promoter led to superior acute and memory CD8 T-cell responses compared to those to p7.5- and pS-driven OVA. Moreover, OVA expressed under the control of pHyb replaced the MVA-derived B8R protein as the immunodominant CD8 T-cell antigen after three or more immunizations. This is the first demonstration of an immediate-early neoantigen expressed by a poxviral vector resulting in superior induction of neoantigen-specific CD8 T-cell responses

NLRC4 Inflammasome-Driven Immunogenicity of a Recombinant MVA Mucosal Vaccine Encoding Flagellin

Bacterial flagellin enhances innate and adaptive immune responses and is considered a promising adjuvant for the development of vaccines against infectious diseases and cancer. Antigen-presenting cells recognize flagellin with the extracellular TLR5 and the intracellular NLRC4 inflammasome-mediated pathway. The detailed cooperation of these innate pathways in the induction of the adaptive immune response following intranasal (i.n.) administration of a recombinant modified vaccinia virus Ankara (rMVA) vaccine encoding flagellin (rMVA-flagellin) is not known. rMVA-flagellin induced enhanced secretion of mucosal IL-1β and TNF-α resulting in elevated CTL and IgG2c antibody responses. Importantly, mucosal IgA responses were also significantly enhanced in both bronchoalveolar (BAL) and intestinal lavages accompanied by the increased migration of CD8+ T cells to the mesenteric lymph nodes (MLN). Nlrc4−/− rMVA-flagellin-immunized mice failed to enhance pulmonary CTL responses, IgG2c was lower, and IgA levels in the BAL or intestinal lavages were similar as those of control mice. Our results show the favorable adjuvant effect of rMVA-flagellin in the lung as well as the intestinal mucosa following i.n. administration with NLRC4 as the essential driver of this promising mucosal vaccine concept

Intravenous but not intranasal post-exposure MVA immunization induces strong systemic cytokine production.

<p>C57BL/6 mice were infected intranasally with 3×10⁴ TCID₅₀ of ECTV. Mice were left untreated or immunized after 3 days with 5×10⁷ TCID₅₀ of MVA either intravenously or intranasally. 6 hours after MVA immunization mice were bled and serum cytokine levels were determined using a bead-based detection assay. Data are means ± SD of 5 mice per group and are representative of three similar experiments. (n.d.  =  not detectable)</p

Intravenous post-exposure MVA immunization induces stronger antigen-specific CD8 T cell response than intranasal immunization.

<p>C57BL/6 mice were infected intranasally with 3×10⁴ TCID₅₀ of ECTV. Mice were left untreated or immunized after 3 days with 5×10⁷ TCID₅₀ of MVA either intravenously or intranasally. 5 days after MVA immunization spleens were removed, stained and analyzed by FACS. (A) Representative contour plots are shown for CD8 T cells (CD3⁺ CD8⁺). Numbers show B8-specific cells as percentage of CD8 T cells. (B) The frequencies of B8-specific CD8 T cells are shown for each group as means ± SD of 5 mice per group. Two more experiments showed similar results.</p

Geometric mean titers of antibodies.

<p>C57BL/6 mice were infected with 3×10⁴ TCID₅₀ of ECTV intranasally. Mice were left untreated or immunized three days later with 5×10⁷ TCID₅₀ of MVA either intravenously or intranasally. Anti-MVA IgG titers were calculated by linear regression and defined as the serum dilution that resulted in an optical density of 0.30. Sera with OD value below 0.3 set to titer of 1 (negative). Geometric mean of neutralizing antibody titers by PRNT were determined as serum dilution able to neutralize 50% of the virus. Similar results were obtained in a separate experiment.</p><p>n.a. not applicable (mouse was dead or bleeding was not possible).</p

Biphasic mechanisms of post-exposure MVA immunization.

<p>In an early phase after ECTV infection (around day 8–20) NK cells, CD4 T cells and antibodies are needed to confer protection after post-exposure MVA immunization. The requirement for IgG can be partly overcome by the production of high amounts of IgM. In a second phase starting around day 21 CD8 T cells become essential. (dpi  =  day post infection)</p

Innate and adaptive immune mechanisms are required for post-exposure protection provided by intravenous MVA immunization.

<p>Mice were infected intranasally with 3×10⁴ TCID₅₀ of ECTV. (A) Wild-type C57BL/6 mice were left untreated (wt control) or immunized (wt MVA) 3 days later with 5×10⁷ TCID₅₀ of MVA intravenously. (B, C and D) All mice were immunized 3 days after ECTV infection with 5×10⁷ TCID₅₀ of MVA intravenously. Survival was monitored for 32 days. The experiments were performed with the indicated number of mice, and data show the cumulative results of 10 experiments.</p

Intravenous post-exposure MVA immunization provides better protection than intranasal immunization.

<p>C57BL/6 mice were infected intranasally with 3×10⁴ TCID₅₀ of ECTV. Mice were left untreated or immunized after 2 (A), 3 (B) or 5 (C) days with 5×10⁷ TCID₅₀ of MVA either intravenously or intranasally. Note, in C no i.n. group was included. Survival was monitored for 32 days. The experiments were performed with 3–5 mice/group. Experiments for A were performed twice, for C once and B shows one exemplary experiment out of three.</p