A SARS-CoV-2 RBD vaccine fused to the chemokine MIP-3α elicits sustained murine antibody responses over 12 months and enhanced lung T-cell responses.

Background: Previous studies have demonstrated enhanced efficacy of vaccine formulations that incorporate the chemokine macrophage inflammatory protein 3α (MIP-3α) to direct vaccine antigens to immature dendritic cells. To address the reduction in vaccine efficacy associated with a mutation in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants, we have examined the ability of receptor-binding domain vaccines incorporating MIP-3α to sustain higher concentrations of antibody when administered intramuscularly (IM) and to more effectively elicit lung T-cell responses when administered intranasally (IN). Methods: BALB/c mice aged 6-8 weeks were immunized intramuscularly or intranasally with DNA vaccine constructs consisting of the SARS-CoV-2 receptor-binding domain alone or fused to the chemokine MIP-3α. In a small-scale (n = 3/group) experiment, mice immunized IM with electroporation were followed up for serum antibody concentrations over a period of 1 year and for bronchoalveolar antibody levels at the termination of the study. Following IN immunization with unencapsulated plasmid DNA (n = 6/group), mice were evaluated at 11 weeks for serum antibody concentrations, quantities of T cells in the lungs, and IFN-γ- and TNF-α-expressing antigen-specific T cells in the lungs and spleen. Results: At 12 months postprimary vaccination, recipients of the IM vaccine incorporating MIP-3α had significantly, approximately threefold, higher serum antibody concentrations than recipients of the vaccine not incorporating MIP-3α. The area-under-the-curve analyses of the 12-month observation interval demonstrated significantly greater antibody concentrations over time in recipients of the MIP-3α vaccine formulation. At 12 months postprimary immunization, only recipients of the fusion vaccine had concentrations of serum-neutralizing activity deemed to be effective. After intranasal immunization, only recipients of the MIP-3α vaccine formulations developed T-cell responses in the lungs significantly above those of PBS controls. Low levels of serum antibody responses were obtained following IN immunization. Conclusion: Although requiring separate IM and IN immunizations for optimal immunization, incorporating MIP-3α in a SARS-CoV-2 vaccine construct demonstrated the potential of a stable and easily produced vaccine formulation to provide the extended antibody and T-cell responses that may be required for protection in the setting of emerging SARS-CoV-2 variants. Without electroporation, simple, uncoated plasmid DNA incorporating MIP-3α administered intranasally elicited lung T-cell responses.

An intranasal stringent response vaccine targeting dendritic cells as a novel adjunctive therapy against tuberculosis.

Lengthy tuberculosis (TB) treatment is required to overcome the ability of a subpopulation of persistent Mycobacterium tuberculosis (Mtb) to remain in a non-replicating, antibiotic-tolerant state characterized by metabolic remodeling, including induction of the RelMtb-mediated stringent response. We developed a novel therapeutic DNA vaccine containing a fusion of the relMtb gene with the gene encoding the immature dendritic cell-targeting chemokine, MIP-3α/CCL20. To augment mucosal immune responses, intranasal delivery was also evaluated. We found that intramuscular delivery of the MIP-3α/relMtb (fusion) vaccine or intranasal delivery of the relMtb (non-fusion) vaccine potentiate isoniazid activity more than intramuscular delivery of the DNA vaccine expressing relMtb alone in a chronic TB mouse model (absolute reduction of Mtb burden: 0.63 log10 and 0.5 log10 colony-forming units, respectively; P=0.0002 and P=0.0052), inducing pronounced Mtb-protective immune signatures. The combined approach involving intranasal delivery of the DNA MIP-3α/relMtb fusion vaccine demonstrated the greatest mycobactericidal activity together with isoniazid when compared to each approach alone (absolute reduction of Mtb burden: 1.13 log10, when compared to the intramuscular vaccine targeting relMtb alone; P<0.0001), as well as robust systemic and local Th1 and Th17 responses. This DNA vaccination strategy may be a promising adjunctive approach combined with standard therapy to shorten curative TB treatment, and also serves as proof of concept for treating other chronic bacterial infections.

IFNα and 5-Aza-2'-deoxycytidine combined with a dendritic-cell targeting DNA vaccine alter tumor immune cell infiltration in the B16F10 melanoma model.

Introduction: DNA vaccines containing a fusion of the gene encoding chemokine MIP-3α (CCL20), the ligand for CCR6 on immature dendritic cells (DCs), to melanoma-associated antigen genes have enhanced anti-tumor immunity and efficacy compared to those lacking the chemokine gene. Previous work has shown that type-I interferon (IFNα or IFN) and 5-Aza-2'-deoxycytidine (5Aza) significantly enhance the therapeutic benefit of DNA vaccines as measured by reduced tumor burden and improved mouse survival. Methods: Here, we explored mouse intratumoral immune correlates underlying the therapeutic benefit of this combination regimen (vaccine, IFN, and 5Aza) as compared to vaccine alone and IFN and 5Aza without vaccine, focusing on chemokine mRNA expression by qRT-PCR and inflammatory cellular infiltration into the tumor microenvironment (TME) by flow cytometry and immunohistochemistry (IHC). Results: The combination group significantly upregulated intratumoral mRNA expression of key immune infiltration chemokines XCL1 and CXCL10. Flow cytometric analyses of tumor suspensions exhibited greater tumor infiltration of CD8+ DCs, CCR7+ DCs, and NK cells in the combination group, as well as reduced levels of myeloid-derived suppressor cells (MDSCs) in vaccinated groups. The mice receiving combination therapy also had greater proportions of effector/memory T-cells (Tem), in addition to showing an enhanced infiltration of Tem and central memory CD8+ T-cells, (Tcm). Tem and Tcm populations both correlated with smaller tumor size. Immunohistochemical analysis of tumors confirmed that CD8+ cells were more abundant overall and especially in the tumor parenchyma with combination therapy. Discussion: Efficient targeting of antigen to immature DCs with a chemokine-fusion vaccine offers a potential alternative approach to classic and dendritic cell-based vaccines. Combining this approach with IFNα and 5Aza treatments significantly improved vaccine efficacy. This treatment creates an environment of increased inflammatory chemokines that facilitates the trafficking of CD8+ DCs, NK cells, and CD8+ T-cells, especially memory cells, while reducing the number of MDSCs. Importantly, in the combination group, CD8+ cells were more able to penetrate the tumor mass in addition to being more numerous. Further analysis of the pathways engaged by our combination therapy is expected to provide additional insights into melanoma pathogenesis and facilitate the development of novel treatment strategies.

Feeding Saccharomyces cerevisiae fermentation products lessens the severity of a viral-bacterial coinfection in preweaned calves.

We have previously reported that supplementation with Saccharomyces cerevisiae fermentation products (SCFP) ameliorates clinical signs and lung pathology following experimental bovine respiratory syncytial virus (BRSV) infection in preweaned dairy calves. The objectives of this study were to determine the effect of SCFP supplementation on the metabolic and endocrine responses, and disease outcome of a viral-bacterial coinfection in preweaned calves. Twenty-seven, 1- to 2-d-old Holstein-Angus cross calves were enrolled in the study; one SCFP calf was removed from the trial during the pre-challenge phase due to complications from nephritis. Calves were assigned to two treatment groups: control or SCFP-treated, base milk replacer with 1 g/d SCFP (Smartcare, soluble formula) and calf starter top dressed with 5 g/d SCFP (NutriTek, insoluble formula). Calves were infected with BRSV on day 21, followed 6 d later by intratracheal inoculation with Pasteurella multocida (PM). Calves were euthanized on day 10 post-viral infection. Calves receiving SCFP had reduced thoracic ultrasonography scores on day 7 post-viral infection (P = 0.03) and a tendency toward reduced scores on day 10 post-viral infection (P = 0.09). Calves receiving SCFP also had less severe lung pathology scores at necropsy (P = 0.06). No differences between treatments were observed in lung viral loads (P = 0.48) or bacterial lung recovery (P = 0.34); however, there was a distinction in the lung location for PM recovery, with PM isolated more frequently from the cranial lobes in SCFP-treated calves, but more frequently from the caudal lobes of control calves. Calves treated with SCFP tended (P = 0.07) to have higher serum IL-6 concentrations following the coinfection. Calves treated with SCFP had lower concentrations of serum nonesterified fatty acids and beta-hydroxybutyric acid compared with controls following experimental challenge (P = 0.03 and P = 0.08, respectively), suggesting metabolic changes favoring growth and development. There were no differences between groups in gene expression of insulin receptor, insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), growth hormone receptor, or haptoglobin in the liver. Results from this study suggest that supplementing with SCFP may moderate the impact of a respiratory viral-bacterial coinfection on preweaned calves through metabolic and immune modifications.