Combinatorial Immunotherapy with Agonistic CD40 Activates Dendritic Cells to Express IL12 and Overcomes PD-1 Resistance.

Checkpoint inhibitors have revolutionized cancer treatment, but resistance remains a significant clinical challenge. Myeloid cells within the tumor microenvironment can modulate checkpoint resistance by either supporting or suppressing adaptive immune responses. Using an anti-PD-1-resistant mouse melanoma model, we show that targeting the myeloid compartment via CD40 activation and CSF1R blockade in combination with anti-PD-1 results in complete tumor regression in a majority of mice. This triple therapy combination was primarily CD40 agonist-driven in the first 24 hours after therapy and showed a similar systemic cytokine profile in human patients as was seen in mice. Functional single-cell cytokine secretion profiling of dendritic cells (DC) using a novel microwell assay identified a CCL22+CCL5+ IL12-secreting DC subset as important early-stage effectors of triple therapy. CD4+ and CD8+ T cells are both critical effectors of treatment, and systems analysis of single-cell RNA sequencing data supported a role for DC-secreted IL12 in priming T-cell activation and recruitment. Finally, we showed that treatment with a novel IL12 mRNA therapeutic alone was sufficient to overcome PD-1 resistance and cause tumor regression. Overall, we conclude that combining myeloid-based innate immune activation and enhancement of adaptive immunity is a viable strategy to overcome anti-PD-1 resistance.

Use of Heterologous Vesiculovirus G Proteins Circumvents the Humoral Anti-envelope Immunity in Lentivector-Based In Vivo Gene Delivery.

Vesicular stomatitis virus Indiana strain glycoprotein (VSVind.G) mediates broad tissue tropism and efficient cellular uptake. Lentiviral vectors (LVs) are particularly promising, as they can efficiently transduce non-dividing cells and facilitate stable genomic transgene integration; therefore, LVs have an enormous untapped potential for gene therapy applications, but the development of humoral and cell-mediated anti-vector responses may restrict their efficacy. We hypothesized that G proteins from different members of the vesiculovirus genus might allow the generation of a panel of serotypically distinct LV pseudotypes with potential for repeated in vivo administration. We found that mice hyperimmunized with VSVind.G were not transduced to any significant degree following intravenous injection of LVs with VSVind.G envelopes, consistent with the thesis that multiple LV administrations would likely be blunted by an adaptive immune response. Excitingly, bioluminescence imaging studies demonstrated that the VSVind-neutralizing response could be evaded by LV pseudotyped with Piry and, to a lesser extent, Cocal virus glycoproteins. Heterologous dosing regimens using viral vectors and oncolytic viruses with Piry and Cocal envelopes could represent a novel strategy to achieve repeated vector-based interventions, unfettered by pre-existing anti-envelope antibodies.

Protection against bubonic and pneumonic plague with a single dose microencapsulated sub-unit vaccine.

Protection against virulent plague challenge by the parenteral and aerosol routes was afforded by a single administration of microencapsulated Caf1 and LcrV antigens from Yersinia pestis in BALB/c mice. Recombinant Caf1 and LcrV were individually encapsulated in polymeric microspheres, to the surface of which additional antigen was adsorbed. The microspheres containing either Caf1 or LcrV were blended and used to immunise mice on a single occasion, by either the intra-nasal or intra-muscular route. Both routes of immunisation induced systemic and local immune responses, with high levels of serum IgG being developed in response to both vaccine antigens. In Elispot assays, secretion of cytokines by spleen and draining lymph node cells was demonstrated, revealing activation of both Th1 and Th2 associated cytokines; and spleen cells from animals immunised by either route were found to proliferate in vitro in response to both vaccine antigens. Virulent challenge experiments demonstrated that non-invasive immunisation by intra-nasal instillation can provide strong systemic and local immune responses and protect against high level challenge. Microencapsulation of these vaccine antigens has the added advantage that controlled release of the antigens occurs in vivo, so that protective immunity can be induced after only a single immunising dose.

Protection against heterologous Burkholderia pseudomallei strains by dendritic cell immunization.

Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacterium which can cause either chronic infections or acute lethal sepsis in infected individuals. The disease is endemic in Southeast Asia and northern Australia, but little is known about the mechanisms of protective immunity to the bacterium. In this study, we have developed a procedure to utilize dendritic cells in combination with CpG oligodeoxynucleotides as a vaccine delivery vector to induce protective immune responses to various strains of B. pseudomallei. Our results show that strong cell-mediated immune responses were generated, while antibody responses, although low, were detectable. Upon virulent challenge with B. pseudomallei strain K96243, NCTC 4845, or 576, animals immunized with dendritic cells that were pulsed with heat-killed K96243 and matured in the presence of CpG 1826 showed significant levels of protection. These results show that a vaccine strategy that actively targets dendritic cells can evoke protective immune responses.

Induction of protective immunity against lethal anthrax challenge with a patch.

BACKGROUND: Transcutaneous immunization (TCI) is a needle-free technique that delivers antigens and adjuvants to potent epidermal immune cells. To address critical unmet needs in biodefense against anthrax, we have designed a novel vaccine delivery system using a dry adhesive patch that simplifies administration and improves tolerability of a subunit anthrax vaccine. METHODS: Mice and rabbits were vaccinated with recombinant protective antigen of Bacillus anthracis and the heat-labile toxin of Escherichia coli. Serologic changes, levels of toxin-neutralizing antibodies (TNAs), and pulmonary and nodal responses were monitored in the mice. A lethal aerosolized B. anthracis challenge model was used in A/J mice, to demonstrate efficacy. RESULTS: The level of systemic immunity and protection induced by TCI was comparable to that induced by intramuscular vaccination, and peak immunity could be achieved with only 2 doses. The addition of adjuvant in the patch induced superior TNA levels, compared with injected vaccination. CONCLUSIONS: Anthrax vaccine patches stimulated robust and functional immune responses that protected against lethal challenge. Demonstration of responses in the lung suggests that a mechanism exists for protection against challenge with aerosolized anthrax spores. A formulated, pressure-sensitive, dry adhesive patch, which is stable and can be manufactured in large scale, elicited comparable immunoglobulin G and TNA responses, suggesting that an anthrax vaccine patch is feasible and should advance into clinical evaluation.