Skin-Grafting and Dendritic Cell "Boosted" Humanized Mouse Models Allow the Pre-Clinical Evaluation of Therapeutic Cancer Vaccines.

Vaccines have been hailed as one of the most remarkable medical advancements in human history, and their potential for treating cancer by generating or expanding anti-tumor T cells has garnered significant interest in recent years. However, the limited efficacy of therapeutic cancer vaccines in clinical trials can be partially attributed to the inadequacy of current preclinical mouse models in recapitulating the complexities of the human immune system. In this study, we developed two innovative humanized mouse models to assess the immunogenicity and therapeutic effectiveness of vaccines targeting human papillomavirus (HPV16) antigens and delivering tumor antigens to human CD141+ dendritic cells (DCs). Both models were based on the transference of human peripheral blood mononuclear cells (PBMCs) into immunocompromised HLA-A*02-NSG mice (NSG-A2), where the use of fresh PBMCs boosted the engraftment of human cells up to 80%. The dynamics of immune cells in the PBMC-hu-NSG-A2 mice demonstrated that T cells constituted the vast majority of engrafted cells, which progressively expanded over time and retained their responsiveness to ex vivo stimulation. Using the PBMC-hu-NSG-A2 system, we generated a hyperplastic skin graft model expressing the HPV16-E7 oncogene. Remarkably, human cells populated the skin grafts, and upon vaccination with a DNA vaccine encoding an HPV16-E6/E7 protein, rapid rejection targeted to the E7-expressing skin was detected, underscoring the capacity of the model to mount a vaccine-specific response. To overcome the decline in DC numbers observed over time in PBMC-hu-NSG-A2 animals, we augmented the abundance of CD141+ DCs, the specific targets of our tailored nanoemulsions (TNEs), by transferring additional autologous PBMCs pre-treated in vitro with the growth factor Flt3-L. The Flt3-L treatment bolstered CD141+ DC numbers, leading to potent antigen-specific CD4+ and CD8+ T cell responses in vivo, which caused the regression of pre-established triple-negative breast cancer and melanoma tumors following CD141+ DC-targeting TNE vaccination. Notably, using HLA-A*02-matching PBMCs for humanizing NSG-A2 mice resulted in a delayed onset of graft-versus-host disease and enhanced the efficacy of the TNE vaccination compared with the parental NSG strain. In conclusion, we successfully established two humanized mouse models that exhibited strong antigen-specific responses and demonstrated tumor regression following vaccination. These models serve as valuable platforms for assessing the efficacy of therapeutic cancer vaccines targeting HPV16-dysplastic skin and diverse tumor antigens specifically delivered to CD141+ DCs.

Repurposing of Commercially Existing Molecular Target Therapies to Boost the Clinical Efficacy of Immune Checkpoint Blockade.

Immune checkpoint blockade (ICB) is now standard of care for several metastatic epithelial cancers and prolongs life expectancy for a significant fraction of patients. A hostile tumor microenvironment (TME) induced by intrinsic oncogenic signaling induces an immunosuppressive niche that protects the tumor cells, limiting the durability and efficacy of ICB therapies. Addition of receptor tyrosine kinase inhibitors (RTKi) as potential modulators of an unfavorable local immune environment has resulted in moderate life expectancy improvement. Though the combination strategy of ICB and RTKi has shown significantly better results compared to individual treatment, the benefits and adverse events are additive whereas synergy of benefit would be preferable. There is therefore a need to investigate the potential of inhibitors other than RTKs to reduce malignant cell survival while enhancing anti-tumor immunity. In the last five years, preclinical studies have focused on using small molecule inhibitors targeting cell cycle and DNA damage regulators such as CDK4/6, CHK1 and poly ADP ribosyl polymerase (PARP) to selectively kill tumor cells and enhance cytotoxic immune responses. This review provides a comprehensive overview of the available drugs that attenuate immunosuppression and overcome hostile TME that could be used to boost FDA-approved ICB efficacy in the near future.

Evolution of Cancer Vaccines-Challenges, Achievements, and Future Directions.

The development of cancer vaccines has been intensively pursued over the past 50 years with modest success. However, recent advancements in the fields of genetics, molecular biology, biochemistry, and immunology have renewed interest in these immunotherapies and allowed the development of promising cancer vaccine candidates. Numerous clinical trials testing the response evoked by tumour antigens, differing in origin and nature, have shed light on the desirable target characteristics capable of inducing strong tumour-specific non-toxic responses with increased potential to bring clinical benefit to patients. Novel delivery methods, ranging from a patient's autologous dendritic cells to liposome nanoparticles, have exponentially increased the abundance and exposure of the antigenic payloads. Furthermore, growing knowledge of the mechanisms by which tumours evade the immune response has led to new approaches to reverse these roadblocks and to re-invigorate previously suppressed anti-tumour surveillance. The use of new drugs in combination with antigen-based therapies is highly targeted and may represent the future of cancer vaccines. In this review, we address the main antigens and delivery methods used to develop cancer vaccines, their clinical outcomes, and the new directions that the vaccine immunotherapy field is taking.

Cytokine/chemokine profiles in squamous cell carcinoma correlate with precancerous and cancerous disease stage.

Actinic Keratosis (AK), Intraepidermal Carcinoma (IEC), and Squamous Cell Carcinoma (SCC) are generally considered to be advancing stages of the same disease spectrum. However, while AK often regress spontaneously, and IEC often regress in response to immune-activating treatments, SCC typically do not regress. Therefore, it is vital to define whether fundamental immunological changes occur during progression to SCC. Here we show that proinflammatory cytokine expression, chemokine expression, and immune cell infiltration density change during progression to SCC. Our findings suggest a switch from predominantly proinflammatory cytokine production to chemokine production is a key feature of progression from precancer to cancer. Together, these observations propose a model that can underpin current research and open new avenues of exploration into the clinical significance of these profiles with respect to immunotherapeutic or other treatment outcomes.

Monocyte-derived dendritic cells enhance protection against secondary influenza challenge by controlling the switch in CD8+ T-cell immunodominance.

Influenza virus infection triggers an increase in the number of monocyte-derived dendritic cells (moDCs) in the respiratory tract, but the role of these cells during antiviral immunity is still unclear. Here we show that during influenza infection, moDCs dominate the late activation of CD8+ T cells and trigger the switch in immunodominance of the CD8+ T-cell response from acidic polymerase specificity to nucleoprotein specificity. Abrogation of monocyte recruitment or depletion of moDCs strongly compromised host resistance to secondary influenza challenge. These findings underscore a novel function of moDCs in the antiviral response to influenza virus, and have important implications for vaccine design.

Mucosal polyinosinic-polycytidylic acid improves protection elicited by replicating influenza vaccines via enhanced dendritic cell function and T cell immunity.

Live-attenuated influenza vaccines (LAIVs) have the potential to generate CD8 T cell immunity that may limit the virulence of an antigenically shifted influenza strain in a population lacking protective Abs. However, current LAIVs exert limited T cell immunity restricted to the vaccine strains. One approach to improve LAIV-induced T cell responses is the use of specific adjuvants to enhance T cell priming by respiratory dendritic cells, but this hypothesis has not been addressed. In this study, we assessed the effect of the TLR3 ligand polyinosinic-polycytidylic acid (poly IC) on CD8 T cell immunity and protection elicited by LAIVs. Mucosal treatment with poly IC shortly after vaccination enhanced respiratory dendritic cell function, CD8 T cell formation, and production of neutralizing Abs. This adjuvant effect of poly IC was dependent on amplification of TLR3 signaling by nonhematopoietic radioresistant cells and enhanced mouse protection to homosubtypic, as well as heterosubtypic, virus challenge. Our findings indicate that mucosal TLR3 ligation may be used to improve CD8 T cell responses to replicating vaccines, which has implications for protection in the absence of pre-existing Ab immunity.

Alphacoronavirus protein 7 modulates host innate immune response.

Innate immune response is the first line of antiviral defense resulting, in most cases, in pathogen clearance with minimal clinical consequences. Viruses have developed diverse strategies to subvert host defense mechanisms and increase their survival. In the transmissible gastroenteritis virus (TGEV) as a model, we previously reported that accessory gene 7 counteracts the host antiviral response by associating with the catalytic subunit of protein phosphatase 1 (PP1c). In the present work, the effect of the absence of gene 7 on the host cell, during infection, was further analyzed by transcriptomic analysis. The pattern of gene expression of cells infected with a recombinant mutant TGEV, lacking gene 7 expression (rTGEV-Δ7), was compared to that of cells infected with the parental virus (rTGEV-wt). Genes involved in the immune response, the interferon response, and inflammation were upregulated during TGEV infection in the absence of gene 7. An exacerbated innate immune response during infection with rTGEV-Δ7 virus was observed both in vitro and in vivo. An increase in macrophage recruitment and activation in lung tissues infected with rTGEV-Δ7 virus was observed compared to cells infected with the parental virus. In summary, the absence of protein 7 both in vitro and in vivo led to increased proinflammatory responses and acute tissue damage after infection. In a porcine animal model, which is immunologically similar to humans, we present a novel example of how viral proteins counteract host antiviral pathways to determine the infection outcome and pathogenesis.

Coronavirus gene 7 counteracts host defenses and modulates virus virulence.

Transmissible gastroenteritis virus (TGEV) genome contains three accessory genes: 3a, 3b and 7. Gene 7 is only present in members of coronavirus genus a1, and encodes a hydrophobic protein of 78 aa. To study gene 7 function, a recombinant TGEV virus lacking gene 7 was engineered (rTGEV-Δ7). Both the mutant and the parental (rTGEV-wt) viruses showed the same growth and viral RNA accumulation kinetics in tissue cultures. Nevertheless, cells infected with rTGEV-Δ7 virus showed an increased cytopathic effect caused by an enhanced apoptosis mediated by caspase activation. Macromolecular synthesis analysis showed that rTGEV-Δ7 virus infection led to host translational shut-off and increased cellular RNA degradation compared with rTGEV-wt infection. An increase of eukaryotic translation initiation factor 2 (eIF2α) phosphorylation and an enhanced nuclease, most likely RNase L, activity were observed in rTGEV-Δ7 virus infected cells. These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response. In protein 7 a conserved sequence motif that potentially mediates binding to protein phosphatase 1 catalytic subunit (PP1c), a key regulator of the cell antiviral defenses, was identified. We postulated that TGEV protein 7 may counteract host antiviral response by its association with PP1c. In fact, pull-down assays demonstrated the interaction between TGEV protein 7, but not a protein 7 mutant lacking PP1c binding motif, with PP1. Moreover, the interaction between protein 7 and PP1 was required, during the infection, for eIF2α dephosphorylation and inhibition of cell RNA degradation. Inoculation of newborn piglets with rTGEV-Δ7 and rTGEV-wt viruses showed that rTGEV-Δ7 virus presented accelerated growth kinetics and pathology compared with the parental virus. Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival. Therefore, the acquisition of gene 7 by the TGEV genome most likely has provided a selective advantage to the virus.

Vectored vaccines to protect against PRRSV.

PRRSV is the causative agent of the most important infectious disease affecting swine herds worldwide, producing great economic losses. Commercially available vaccines are only partially effective in protection against PRRSV. Moreover, modified live vaccines may allow virus shedding, and could revert generating virulent phenotypes. Therefore, new efficient vaccines are required. Vaccines based on recombinant virus genomes (virus vectored vaccines) against PRRSV could represent a safe alternative for the generation of modified live vaccines. In this paper, current vectored vaccines to protect against PRRSV are revised, including those based on pseudorabies virus, poxvirus, adenovirus, and virus replicons. Special attention has been provided to the use of transmissible gastroenteritis virus (TGEV) as vector for the expression of PRRSV antigens. This vector has the capability of expressing high levels of heterologous genes, is a potent interferon-α inducer, and presents antigens in mucosal surfaces, eliciting both secretory and systemic immunity. A TGEV derived vector (rTGEV) was generated, expressing PRRSV wild type or modified GP5 and M proteins, described as the main inducers of neutralizing antibodies and cellular immune response, respectively. Protection experiments showed that vaccinated animals developed a faster and stronger humoral immune response than the non-vaccinated ones. Partial protection in challenged animals was observed, as vaccinated pigs showed decreased lung damage when compared with the non-vaccinated ones. Nevertheless, the level of neutralizing antibodies was low, what may explain the limited protection observed. Several strategies are proposed to improve current rTGEV vectors expressing PRRSV antigens.

Coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription.

Purified nucleocapsid protein (N protein) from transmissible gastroenteritis virus (TGEV) enhanced hammerhead ribozyme self-cleavage and favored nucleic acid annealing, properties that define RNA chaperones, as previously reported. Several TGEV N-protein deletion mutants were expressed in Escherichia coli and purified, and their RNA binding ability and RNA chaperone activity were evaluated. The smallest N-protein domain analyzed with RNA chaperone activity, facilitating DNA and RNA annealing, contained the central unstructured region (amino acids 117 to 268). Interestingly, N protein and its deletion mutants with RNA chaperone activity enhanced template switching in a retrovirus-derived heterologous system, reinforcing the concept that TGEV N protein is an RNA chaperone that could be involved in template switching. This result is in agreement with the observation that in vivo, N protein is not necessary for TGEV replication, but it is required for efficient transcription.

Role of RNA chaperones in virus replication.

RNA molecules are functionally diverse in part due to their extreme structural flexibility that allows rapid regulation by refolding. RNA folding could be a difficult process as often molecules adopt a spatial conformation that is very stable but not biologically functional, named a kinetic trap. RNA chaperones are non-specific RNA binding proteins that help RNA folding by resolving misfolded structures or preventing their formation. There is a large number of viruses whose genome is RNA that allows some evolutionary advantages, such as rapid genome mutation. On the other hand, regions of the viral RNA genomes can adopt different structural conformations, some of them lacking functional relevance and acting as misfolded intermediates. In fact, for an efficient replication, they often require RNA chaperone activities. There is a growing list of RNA chaperones encoded by viruses involved in different steps of the viral cycle. Also, cellular RNA chaperones have been involved in replication of RNA viruses. This review briefly describes RNA chaperone activities and is focused in the roles that viral or cellular nucleic acid chaperones have in RNA virus replication, particularly in those viruses that require discontinuous RNA synthesis.