Development and preclinical evaluation of virus-like particle vaccine against COVID-19 infection.

BACKGROUND: Vaccines that incorporate multiple SARS-CoV-2 antigens can further broaden the breadth of virus-specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS-CoV-2 VLP vaccine that incorporates the four structural proteins of SARS-CoV-2. METHODS: VLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography, and characterized by tunable-resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine. RESULTS: Transiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS-CoV-2. Alum adsorbed, K3-CpG ODN-adjuvanted VLPs elicited high titer anti-S, anti-RBD, anti-N IgG, triggered multifunctional Th1-biased T-cell responses, reduced virus load, and prevented lung pathology upon live virus challenge in vaccinated animals. CONCLUSION: These data suggest that VLPs expressing all four structural protein antigens of SARS-CoV-2 are immunogenic and can protect animals from developing COVID-19 infection following vaccination.

Low-dose natural human interferon-alpha lozenges in the treatment of Behçet's syndrome.

OBJECTIVES: There had been evidence that low-dose local IFN could be beneficial in the management of recurrent oral ulcers (OUs). We investigated the efficacy and collected initial data on the safety of low-dose natural human IFN-alpha administered by the oral mucosal route in Behçet's syndrome (BS) in a placebo controlled, double blind study. METHODS: Eighty-four (59 males and 25 females) patients with BS with mainly skin mucosa disease and a history of recurrent OU for > or = 1 year were studied. When they had at least two OUs with a total diameter of > or = 4 mm, they were randomly allocated to (i) 2000 IFN-alpha IU/day, (ii) 1000 IFN-alpha IU/day and (iii) placebo groups. Subjects were monitored weekly over an initial 4 weeks and bi-weekly for an additional 8 weeks of treatment. OU were counted and measured at each study visit. The primary efficacy end point was the difference in the total ulcer burden at Week 0 compared with that at Week 12. RESULTS: Out of the 84 patients enrolled, 72 completed the trial. There were no statistically significant differences between the treatment arms in terms of the primary endpoint. CONCLUSIONS: Low-dose natural human IFN-alpha did not have beneficial effects on reducing the total ulcer burden among BS patients from Turkey. The study also showed that counting the number of ulcers rather than measuring the size would be adequate in future studies. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00483184, http://www.clinicaltrials.gov/ct2/results?term=NCT00483184.

Targeted delivery of murine IFN-gamma using a recombinant fowlpox virus: NK cell recruitment to regional lymph nodes and priming of tumor-specific host immunity.

Interferon-gamma (IFN-gamma) is a proinflammatory cytokine that also acts as a potent immunomodulatory agent. In this study, a replication-deficient recombinant avian (fowlpox) virus was engineered to express the murine IFN-gamma gene (rF-MuIFN-gamma) with the rationale of delivering concentrated levels of the cytokine to a local tissue microenvironment. Subcutaneous (s.c.) rF-MuIFN-gamma administration resulted in IFN-gamma production that (1) was restricted to the tissue microenvironment of the injection site and (2) was biologically active, as evidenced by a significant increase of class I MHC expression levels in s.c. growing tumors following rF-MuIFN-gamma administration. Infection of a highly tumorigenic murine cell line, MC38, with rF-MuIFN-gamma functioned as an effective tumor cell-based vaccine by protecting mice from the formation of primary tumors and from subsequent tumor challenge. The cell-based vaccine was completely ineffective if mice were vaccinated with MC38 cells either pretreated with rIFN-gamma or infected with the wild-type fowlpox virus (FP-WT). Analysis of the regional lymph nodes draining the site of injection of the rF-MuIFN-gamma-based tumor cell vaccine revealed the presence of tumor-specific cell lysis (CTL) as well as a significant amount of lysis directed at natural killer (NK)-sensitive YAC-1 cells. Flow cytometric analyses coupled with functional assays confirmed the sustained presence of NK1.1(+) cells within those draining lymph nodes for up to 5 days after rF-MuIFN-gamma injection. Mice treated with NK cell-depleting antibodies prior to the injection of the rF-MuIFN-gamma-infected MC38 tumor cells were not protected from primary tumor growth; analysis of the lymph nodes draining the injection site in NK-depleted mice revealed an accompanying loss of the tumor-specific CTL activity. The findings provide evidence that NK cells, known for their contributions to host innate immunity, also provide immunoregulatory signals required for the development of an adaptive immune response, which, in turn, protected vaccinated mice against tumor growth.

Vaccine-based therapy directed against carcinoembryonic antigen demonstrates antitumor activity on spontaneous intestinal tumors in the absence of autoimmunity.

By virtue of its tissue-specific expression, carcinoembryonic antigen (CEA) is an important self, tumor-associated antigen, which is expressed by different human adenocarcinomas and also serves as a target for active-specific immunotherapy. Similar to humans, CEA expression in mice transgenic for the human CEA gene (CEA.Tg) occurs predominantly along the gastrointestinal tract. CEA.Tg mice were crossed with mice bearing a mutation in the Apc gene (MIN mice), and the CEA.Tg/MIN progeny developed multiple intestinal neoplasms, which overexpress CEA to levels that are reminiscent of those reported for tubulovillous intestinal adenomas from patients. CEA.Tg/MIN mice were vaccinated with an aggressive diversified prime/boost vaccine regimen: (a) a primary vaccine consisting of recombinant vaccinia virus-expressing CEA and a triad of costimulatory molecules (TRICOM): B7.1, ICAM-1, and LFA-3 (rV-CEA-TRICOM); and (b) a booster vaccine using CEA-TRICOM in a recombinant avipox (fowlpox) virus (rF-CEA-TRICOM). Granulocyte/macrophage colony-stimulating factor was administered as a biological adjuvant with all vaccinations, either as a recombinant protein (with rV-CEA-TRICOM) or as a recombinant avipox virus (with rF-CEA-TRICOM). That vaccine regimen generated strong CEA-specific host immune responses in CEA.Tg/MIN mice, which resulted in (a) a delayed onset of adult anemia and weight loss, (b) a significant reduction in the number of intestinal tumors, and (c) improved overall survival. No evidence of autoimmunity directed against normal tissues expressing CEA was observed in mice in which the CEA-based vaccine significantly reduced intestinal tumor load. The CEA.Tg/MIN mice present a clinically relevant model in which different CEA-based vaccine strategies can be tested on the spontaneous onset of intestinal tumorigenesis.

Combination of a poxvirus-based vaccine with a cyclooxygenase-2 inhibitor (celecoxib) elicits antitumor immunity and long-term survival in CEA.Tg/MIN mice.

The present study was designed to determine whether: (a) chronic administration of dietary celecoxib (Celebrex), a potent nonsteroidal anti-inflammatory drug, which targets the cyclooxygenase-2 (COX-2) enzyme, negatively impacts host immunity; and (b) celecoxib can be coupled with a poxvirus-based vaccine to impact tumor burden in a murine tumor model of spontaneous adenomatous polyposis coli. Naive mice fed the celecoxib-supplemented diets developed eosinophilia with lowered plasma prostaglandin E(2) levels and reduced COX-2 mRNA expression levels in their splenic T cells. Responses of splenic T, B, and natural killer cells to broad-based and antigen-specific stimuli were, for the most part, unchanged in those mice as well as COX-2 knockout mice; exceptions included: (a) reduced IFN-gamma production by concanavalin A- or antigen-stimulated T cells; and (b) heightened lipopolysaccharide response of naive B cells from mice fed a diet supplemented with 1000 ppm of celecoxib. When transgenic mice that express the human carcinoembryonic antigen (CEA) gene (CEA transgenic) were bred with mice bearing a mutation in the Apc(Delta850) gene (multiple intestinal neoplasia mice), the progeny (CEA transgenic/multiple intestinal neoplasia) spontaneously develop multiple intestinal neoplasms that overexpress CEA and COX-2. Beginning at 30 days of age, the administration of a diversified prime/boost recombinant CEA-poxvirus-based vaccine regimen or celecoxib (1000 ppm)-supplemented diet reduced the number of intestinal neoplasms by 54% and 65%, respectively. Combining the CEA-based vaccine with the celecoxib-supplemented diet reduced tumor burden by 95% and significantly improved overall long-term survival. Both tumor reduction and improved overall survival were achieved without any evidence of autoimmunity directed at CEA-expressing or other normal tissues. Celecoxib is prescribed for the treatment of familial adenomatous polyposis in humans, and the CEA-based vaccines have been well tolerated and capable of eliciting anti-CEA host immune responses in early clinical studies. The results suggest that the administration of a recombinant poxvirus-based vaccine is compatible with celecoxib, and this combined chemoimmuno-based approach might lead to an additive therapeutic antitumor benefit not only in patients diagnosed with familial adenomatous polyposis but, perhaps, in other preventive settings in which COX-2 overexpression is associated with progression from premalignancy to neoplasia.

Mouse models expressing human carcinoembryonic antigen (CEA) as a transgene: evaluation of CEA-based cancer vaccines.

In recent years, investigators have carried out several studies designed to evaluate whether human tumor-associated antigens might be exploited as targets for active specific immunotherapy, specifically human cancer vaccines. Not too long ago such an approach would have been met with considerable skepticism because the immune system was believed to be a rigid discriminator between self and non-self which, in turn, protected the host from a variety of pathogens. That viewpoint has been challenged in recent years by a series of studies indicating that antigenic determinants of self have not induced absolute host immune tolerance. Moreover, under specific conditions that evoke danger signals, peptides from self-antigen can be processed by the antigen-presenting cellular machinery, loaded onto the major histocompatibility antigen groove to serve as targets for immune intervention. Those findings provide the rationale to investigate a wide range of tumor-associated antigens, including differentiation antigens, oncogenes, and tumor suppressor genes as possible immune-based targets. One of those tumor-associated antigens is the carcinoembryonic antigen (CEA). Described almost 40 years ago, CEA is a M(r) 180-200,000 oncofetal antigen that is one of the more widely studied human tumor-associated antigens. This review will provide: (i) a brief overview of the CEA gene family, (ii) a summary of early preclinical findings on overcoming immune tolerance to CEA, and (iii) the rationale to develop mouse models which spontaneously develop gastrointestinal tumors and express the CEA transgene. Those models have been used extensively in the study of overcoming host immune tolerance to CEA, a self, tumor-associated antigen, and the experimental findings have served as the rationale for the design of early clinical trials to evaluate CEA-based cancer vaccines.

Enhanced expression of lymphotactin by CD8+ T cells is selectively induced by enhancer agonist peptides of tumor-associated antigens.

Human tumor-associated antigens (TAAs) are weak immunogens. One strategy for increasing the immunogenicity of TAAs is to generate altered peptide ligands. In the studies reported here, microarray technology has been used to compare gene expression profiles of a human T cell line that was derived from the peripheral blood of a cancer patient vaccinated with a carcinoembryonic antigen (CEA)-based vaccine. We compared the gene expression profiles of this CEA-specific CD8 T cell line when (a) stimulated with the native peptide used to derive this line vs. no peptide, and (b) stimulated with its TCR enhancer agonist epitope vs. no peptide. The results demonstrate that the effect on the T cell line, when stimulated with the agonist peptide, is not an enhanced quantitative expression of the same genes or gene sets induced by the native peptide, but is rather a nearly reciprocal upregulation of different gene sets. The gene for the chemokine lymphotactin, which was overexpressed only in T cells stimulated with the agonist peptide, stood out above all others. This finding was extended using other T cell lines, and another set of agonist and native peptides from another TAA. ELISPOT and ELISA assays for lymphotactin confirmed and extended these findings. These studies suggest a potential role for lymphotactin in the T-cell activation processes and subsequent anti-tumor events.