Immunogenicity in mice and rhesus monkeys vaccinated with recombinant vaccinia virus expressing bivalent E7E6 fusion proteins from human papillomavirus types 16 and 18

<p>Abstract</p> <p>Background</p> <p>Persistent infection with high-risk human papillomavirus (HPV) is a predominant cause of cervical cancer, and HPV16 and HPV18 occur in 50% and 20% of cervical cancer cases, respectively. The viral oncogenes E6 and E7 are constitutively expressed by HPV-associated tumour cells and can therefore be used as target antigens for immunotherapy. In this study, we constructed a recombinant vaccinia virus co-expressing the HPV16/18 E7E6 fusion proteins (rVVJ16/18E7E6) for use as a therapeutic vaccine for the treatment of HPV16⁺and HPV18⁺cancers.</p> <p>Methods</p> <p>We constructed a bivalent recombinant vaccinia virus expressing modified E7E6 fusion proteins of HPV type 16 and 18 (rVVJ16/18E7E6) based on the vaccinia virus Tiantan strain. We then defined the cellular immune responses to the virus in mice and rhesus monkeys and assessed antitumour efficacy of these responses in mice using the TC-1 tumour challenge model.</p> <p>Results</p> <p>Our data demonstrated that rVVJ16/18E7E6 was able to elicit varying levels of CD8⁺T cell immune responses and lysis of target cells in mice in response to peptides HPV16E7_49-57and HPV18E6_67-75. Furthermore, the virus was also able to induce anti-tumour responses in the HPV16⁺TC-1 tumour challenge model, including partial protection (30-40%) and delayed tumour appearance. In addition, the virus was able to induce immune responses in rhesus monkeys.</p> <p>Conclusions</p> <p>The recombinant vaccinia virus rVVJ16/18E7E6 can generate clear and significant cellular immunity in both mice and rhesus monkeys. These data provide a basis for the use of this recombinant virus as a potential vaccine candidate for further study.</p

Molecular biology of human enteric caliciviruses

At the start of this thesis the genomes of two SRSVs Southampton virus (SV) and Norwalk virus (NV) have been characterised. However, sequence comparison between two SRSVs and animal caliciviruses suggested that the published SV and NV genome sequences may be incomplete as they both lack conserved motifs at the 5' termini of their respective genomes. This led to a further attempt to define the SV genomic 5' terminal sequence using RT-PCR and homopolymer tailing. An additional sequence of 12 nucleotides was identified which significantly changed the genome organisation by extending the first large open reading frame (ORF) by 51 amino acids. The 5' terminal bases pGpT and the presence of conserved genome and putative subgenomic RNA terminal motifs are now prominent features shared between the SV and the animal caliciviruses RHDV and FCV.A genomic clone of SV was constructed, sequenced and used to study translation of the genomic RNA in vitro. Three major translation products of 113, 48 and 41 kDa were identified in a coupled transcription-translation system.Classic human enteric caliciviruses (HuCVs) have a distinctive morphology and are primarily associated with paediatric acute gastro-enteritis. Although morphologically distinct from the small round structured viruses (SRSVs), the classic HuCVs are thought to be closely related and were anticipated to have a similar genome organisation. The complete genome sequence of a classic HuCV (Manchester virus) was determined. The RNA genome (7266 nt) is smaller than the genome of SRSVs from the two genetic groups and has a unique arrangement of open reading frames.A full-length Manchester virus cDNA clone was constructed and polyprotein processing was investigated in vitro. The major translation products of approximately 38kDa and 28kDa were observed which could represent 2C helicase and N terminal peptide respectively.</p

Immune‐check blocking combination multiple cytokines shown curative potential in mice tumor model

Abstract Objective In order to ensure the stable transcription of target genes, we constructed a eukaryotic high expression vector carrying an immune‐check inhibitor PD‐1v and a variety of cytokines, and studied their effects on activating immune response to inhibit tumor growth. Methods A novel eukaryotic expression plasmid vector named pT7AMPCE containing T7RNA polymerase, T7 promoter, internal ribosome entry site (IRES), and poly A tailing signal was constructed by T4 DNA ligase, on which homologous recombination was used to clone and construct the vector carrying PD‐1v, IL‐2/15, IL‐12, GM‐CSF, and GFP. In vitro transfection of CT26 cells was performed, and the protein expression of PD‐1v, IL‐12 and GM‐CSF was detected by Western blot and ELISA after 48 h. Mice were subcutaneously inoculated with CT26‐IRFP tumor cells in the rib abdomen, and the tumor tissues were injected with PD‐1v, IL‐2/15, IL‐12, and GM‐CSF recombinant plasmids for treatment during the experimental period. The efficacy of the treatment was evaluated by assay tumor size and survival time of tumor‐bearing mice during the experiment. Expression levels of IFN‐γ, TNF, IL‐4, IL‐2, and IL‐5 in mouse blood were measured using the CBA method. Tumor tissues were extracted and immune cell infiltration in tumor tissues was detected by HE staining and the IHC method. Results The recombinant plasmids carrying PD‐1v, IL‐2/15, IL‐12, and GM‐CSF were successfully constructed, and the Western blot and ELISA results showed that PD‐1v, IL‐12, and GM‐CSF were expressed in the supernatant of CT26 cells 48 h after in vitro cell transfection. The combined application of PD‐1v, IL‐2/15, IL‐12, and GM‐CSF recombinant plasmids significantly inhibited tumor growth in mice, and the tumor growth rate was significantly lower than that in the blank control group and GFP plasmid control group (p < 0.05). Cytometric bead array data suggested that the combination of PD‐1v and various cytokines can effectively activate immune cells. HE and IHC analysis revealed plenty of immune cell infiltrates in the tumor tissue, and a large proportion of tumor cells showed the necrotic phenotype in the combination treatment group. Conclusion The combination of immune check blockade and multiple cytokine therapy can significantly activate the body's immune response and inhibit tumor growth

Exploring immune evasion of SARS-CoV-2 variants using a pseudotyped system

In the United States, coronavirus disease 2019 (COVID-19) cases have consistently been linked to the prevailing variant XBB.1.5 of SARS-CoV-2 since late 2022. A system has been developed for producing and infecting cells with a pseudovirus (PsV) of SARS-CoV-2 to investigate the infection in a Biosafety Level 2 (BSL-2) laboratory. This system utilizes a lentiviral vector carrying ZsGreen1 and Firefly luciferase (Fluc) dual reporter genes, facilitating the analysis of experimental results. In addition, we have created a panel of PsV variants that depict both previous and presently circulating mutations found in circulating SARS-CoV-2 strains. A series of PsVs includes the prototype SARS-CoV-2, Delta B.1.617.2, BA.5, XBB.1, and XBB.1.5. To facilitate the study of infections caused by different variants of SARS-CoV-2 PsV, we have developed a HEK-293T cell line expressing mCherry and human angiotensin converting enzyme 2 (ACE2). To validate whether different SARS-CoV-2 PsV variants can be used for neutralization assays, we employed serum from rats immunized with the PF-D-Trimer protein vaccine to investigate its inhibitory effect on the infectivity of various SARS-CoV-2 PsV variants. According to our observations, the XBB variant, particularly XBB.1.5, exhibits stronger immune evasion capabilities than the prototype SARS-CoV-2, Delta B.1.617.2, and BA.5 PsV variants. Hence, utilizing the neutralization test, this study has the capability to forecast the effectiveness in preventing future SARS-CoV-2 variants infections

Intratumoral OH2, an oncolytic herpes simplex virus 2, in patients with advanced solid tumors: a multicenter, phase I/II clinical trial

Background OH2 is a genetically engineered oncolytic herpes simplex virus type 2 designed to selectively amplify in tumor cells and express granulocyte-macrophage colony-stimulating factor to enhance antitumor immune responses. We investigated the safety, tolerability and antitumor activity of OH2 as single agent or in combination with HX008, an anti-programmed cell death protein 1 antibody, in patients with advanced solid tumors.Methods In this multicenter, phase I/II trial, we enrolled patients with standard treatment-refractory advanced solid tumors who have injectable lesions. In phase I, patients received intratumoral injection of OH2 at escalating doses (106, 107 and 108CCID50/mL) as single agent or with fixed-dose HX008. The recommended doses were then expanded in phase II. Primary endpoints were safety and tolerability defined by the maximum-tolerated dose and dose-limiting toxicities (DLTs) in phase I, and antitumor activity assessed per Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) and immune-RECIST in phase II.Results Between April 17, 2019 and September 22, 2020, 54 patients with metastatic cancers were enrolled. Forty patients were treated with single agent OH2, and 14 with OH2 plus HX008. No DLTs were reported with single agent OH2 in phase I. Four patients, having metastatic mismatch repair-proficient rectal cancer or metastatic esophageal cancer, achieved immune-partial response, with two from the single agent cohort and two from the combination cohort. The duration of response were 11.25+ and 14.03+ months for the two responders treated with single agent OH2, and 1.38+ and 2.56+ months for the two responders in the combination cohort. The most common treatment-related adverse event (TRAE) with single agent OH2 was fever (n=18, 45.0%). All TRAEs were of grade 1–2, except one case of grade 3 fever in the 108CCID50/mL group. No treatment-related serious AEs occurred. Single agent OH2 induced alterations in the tumor microenvironment, with clear increases in CD3+ and CD8+ cell density and programmed death-ligand 1 expression in the patients’ post-treatment biopsies relative to baseline.Conclusions Intratumoral injection of OH2 was well-tolerated, and demonstrated durable antitumor activity in patients with metastatic esophageal and rectal cancer. Further clinical development of OH2 as single agent or with immune checkpoint inhibitors in selected tumor types is warranted

A novel oncolytic herpes simplex virus type 2 has potent anti-tumor activity.

Oncolytic viruses are promising treatments for many kinds of solid tumors. In this study, we constructed a novel oncolytic herpes simplex virus type 2: oHSV2. We investigated the cytopathic effects of oHSV2 in vitro and tested its antitumor efficacy in a 4T1 breast cancer model. We compared its effect on the cell cycle and its immunologic impact with the traditional chemotherapeutic agent doxorubicin. In vitro data showed that oHSV2 infected most of the human and murine tumor cell lines and was highly oncolytic. oHSV2 infected and killed 4T1 tumor cells independent of their cell cycle phase, whereas doxorubicin mainly blocked cells that were in S and G2/M phase. In vivo study showed that both oHSV2 and doxorubicin had an antitumor effect, though the former was less toxic. oHSV2 treatment alone not only slowed down the growth of tumors without causing weight loss but also induced an elevation of NK cells and mild decrease of Tregs in spleen. In addition, combination therapy of doxorubicin followed by oHSV2 increased survival with weight loss than oHSV2 alone. The data showed that the oncolytic activity of oHSV2 was similar to oHSV1 in cell lines examined and in vivo. Therefore, we concluded that our virus is a safe and effective therapeutic agent for 4T1 breast cancer and that the sequential use of doxorubicin followed by oHSV2 could improve antitumor activity without enhancing doxorubicin's toxicity

The oncolytic effect of oHSV2 on 4T1 tumor cells is independent of the cell cycle, but oHSV2 increases the NK ratio in vivo.

<p>A) An cell cycle assay as described in the Methods section. Representative images of flow cytometry from the different groups are depicted. B) 4T1 cells were treated with oHSV2 or different doses of DOX for 24h. Cell cycle specificity analysis was performed using flow cytometry. C) The percentage of NK and Treg cells after oHSV2 or DOX treatment was assayed. Statistical analysis was performed using an unpaired Student’s t test: *, p<0.05; **, p<0.01; and ***, p<0.001.</p