Development of a multiplex droplet digital PCR assay for detection of enterovirus, parechovirus, herpes simplex virus 1 and 2 simultaneously for diagnosis of viral CNS infections.

BACKGROUND: Enterovirus (EV), parechovirus (HPeV), herpes simplex virus 1 and 2 (HSV1/2) are common viruses leading to viral central nervous system (CNS) infections which are increasingly predominant but exhibit deficiency in definite pathogen diagnosis with gold-standard quantitative PCR method. Previous studies have shown that droplet digital PCR (ddPCR) has great potential in pathogen detection and quantification, especially in low concentration samples. METHODS: Targeting four common viruses of EV, HPeV, HSV1, and HSV2 in cerebrospinal fluid (CSF), we developed a multiplex ddPCR assay using probe ratio-based multiplexing strategy, analyzed the performance, and evaluated it in 97 CSF samples collected from patients with suspected viral CNS infections on a two-channel ddPCR detection system. RESULTS: The four viruses were clearly distinguished by their corresponding fluorescence amplitude. The limits of detection for EV, HPeV, HSV1, and HSV2 were 5, 10, 5, and 10 copies per reaction, respectively. The dynamic range was at least four orders of magnitude spanning from 2000 to 2 copies per reaction. The results of 97 tested clinical CSF specimens were identical to those deduced from qPCR/qRT-PCR assays using commercial kits. CONCLUSION: The multiplex ddPCR assay was demonstrated to be an accurate and robust method which could detect EV, HPeV, HSV1, and HSV2 simultaneously. It provides a useful tool for clinical diagnosis and disease monitoring of viral CNS infections.

Antibodies to Crucial Epitopes on HSV-2 Glycoprotein D as a Guide to Dosing an mRNA Genital Herpes Vaccine.

The toxicity of mRNA-lipid nanoparticle (LNP) vaccines depends on the total mRNA-LNP dose. We established that the maximum tolerated dose of our trivalent mRNA-LNP genital herpes vaccine was 10 µg/immunization in mice. We then evaluated one of the mRNAs, gD2 mRNA-LNP, to determine how much of the 10 µg total dose to assign to this immunogen. We immunized mice with 0.3, 1.0, 3.0, or 10 µg of gD2 mRNA-LNP and measured serum IgG ELISA, neutralizing antibodies, and antibodies to six crucial gD2 epitopes involved in virus entry and spread. Antibodies to crucial gD2 epitopes peaked at 1 µg, while ELISA and neutralizing titers continued to increase at higher doses. The epitope results suggested no immunologic benefit above 1 µg of gD2 mRNA-LNP, while ELISA and neutralizing titers indicated higher doses may be useful. We challenged the gD2 mRNA-immunized mice intravaginally with HSV-2. The 1-µg dose provided total protection, confirming the epitope studies, and supported assigning less than one-third of the trivalent vaccine maximum dose of 10 µg to gD2 mRNA-LNP. Epitope mapping as performed in mice can also be accomplished in phase 1 human trials to help select the optimum dose of each immunogen in a multivalent vaccine.

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.