Immunogenicity and Efficacy of TNX-1800, A Live Virus Recombinant Poxvirus Vaccine Candidate, against SARS-CoV-2 Challenge in Nonhuman Primates.

TNX-1800 is a synthetically derived live recombinant chimeric horsepox virus (rcHPXV) vaccine candidate expressing Wuhan SARS-CoV-2 spike (S) protein. The primary objective of this study was to evaluate the immunogenicity and efficacy of TNX-1800 in two nonhuman primate species challenged with USA-WA1/2020 SARS-CoV-2. TNX-1800 vaccination was well tolerated with no serious adverse events or significant changes in clinical parameters. A single dose of TNX-1800 generated humoral responses in African Green Monkeys and Cynomolgus Macaques, as measured by the total binding of anti-SARS-CoV-2 S IgG and neutralizing antibody titers against the USA-WA1/2020 strain. In addition, a single dose of TNX-1800 induced an interferon-gamma (IFN-γ)-mediated T-cell response in Cynomolgus Macaques. Following challenge with SARS-CoV-2, African Green and Cynomolgus Macaques exhibited rapid clearance of virus in the upper and lower respiratory tract. Future studies will assess the efficacy of TNX-1800 against newly emerging variants and demonstrate its safety in humans.

Development of a rapid image-based high-content imaging screening assay to evaluate therapeutic antibodies against the monkeypox virus.

Antibody-based therapy is emerging as a critical therapeutic countermeasure to treat acute viral infections by offering rapid protection against clinical disease. The advancements in structural biology made it feasible to rationalize monoclonal antibodies (mAbs) by identifying key and, possibly, neutralizing epitopes of viral proteins for therapeutic purposes. A critical component in assessing mAbs during pandemics requires the development of rapid but detailed methods to detect and quantitate the neutralization activity. In this study, we developed and optimized two high-content image (HCI)-based assays: one to detect viral proteins by staining and the second to quantify cytopathic viral effects by a label-free phenotypic assay. These assays were employed to screen for therapeutic antibodies against the monkeypox virus (MPXV) using surrogate poxviruses such as vaccinia virus (VACV). Plaque-based neutralization results confirmed the HCI data. The phenotypic assay found pox virus-induced syncytia formation in various cells, and we were able to quantitate and use this phenotype to screen mAbs. The HCI identified several potent VACV-neutralizing antibodies that showed in vitro efficacy against both clades of MPXV. In addition, a combination study of ST-246/tecovirimat/TPOXX a single neutralizing antibody Ab-40, showed synergistic activity against VACV in an in-vitro neutralization assay. This rapid high-content method utilizing state-of-the-art technologies enabled the evaluation of hundreds of mAbs quickly to identify several potent anti-MPXV neutralizing mAbs for further development.

Co-administration of tecovirimat and ACAM2000™ in non-human primates: Effect of tecovirimat treatment on ACAM2000 immunogenicity and efficacy versus lethal monkeypox virus challenge.

Naturally occurring smallpox has been eradicated but research stocks of variola virus (VARV), the causative agent of smallpox, still exist in secure laboratories. Clandestine stores of the virus or resurrection of VARV via synthetic biology are possible and have led to concerns that VARV could be used as a biological weapon. The US government has prepared for such an event by stockpiling smallpox vaccines and TPOXX®, SIGA Technologies' smallpox antiviral drug. While vaccination is effective as a pre-exposure prophylaxis, protection is limited when administered following exposure. Safety concerns preclude general use of the vaccine unless there is a smallpox outbreak. TPOXX is approved by the FDA for use after confirmed diagnosis of smallpox disease. Tecovirimat, the active pharmaceutical ingredient in TPOXX, targets a highly conserved orthopoxviral protein, inhibiting long-range dissemination of virus. Although indications for use of the vaccine and TPOXX do not overlap, concomitant use is possible, especially if the TPOXX indication is expanded to include post-exposure prophylaxis. It is therefore important to understand how vaccine and TPOXX may interact. In studies presented here, monkeys were vaccinated with the ACAM2000TM live attenuated smallpox vaccine and concomitantly treated with tecovirimat or placebo. Immune responses to the vaccine and protective efficacy versus a lethal monkeypox virus (MPXV) challenge were evaluated. In two studies, primary and anamnestic humoral immune responses were similar regardless of tecovirimat treatment while the third study showed reduction in vaccine elicited humoral immunity. Following lethal MPXV challenge, all (12 of 12) vaccinated/placebo treated animals survived, and 12 of 13 vaccinated/tecovirimat treated animals survived. Clinical signs of disease were elevated in tecovirimat treated animals compared to placebo treated animals. This suggests that TPOXX may affect the immunogenicity of ACAM2000 if administered concomitantly. These studies may inform on how vaccine and TPOXX are used during a smallpox outbreak.

Construction and characterization of Marek's disease viruses having green fluorescent protein expression tied directly or indirectly to phosphoprotein 38 expression.

Marek's disease (MD) is caused by Marek's disease virus (MDV), a highly cell-associated alphaherpesvirus. MD is primarily characterized by lymphocyte infiltration of the nerves and the development of lymphomas in visceral organs, muscle, and skin. MDV encodes two phosphoproteins, pp24 and pp38, that are highly expressed during lytic infection. These proteins were initially identified in MDV-induced tumors but are now known to be linked primarily to MDV lytic infection. Despite the recent characterization of a pp38 deletion mutant MDV, the functions of these phosphoproteins remain unknown. The goal of this work was to construct recombinant MDVs having direct fusions of a marker gene, the green fluorescent protein (GFP), to pp38 in order to study the expression patterns and localization of this protein during stages of MDV infection. We report the construction of two recombinant viruses, one having the enhanced green fluorescent protein (eGFP) fused in-frame to the pp38 open reading frame (ORF) (RB1Bpp38/eGFP) and the other having soluble-modified GFP (smGFP) downstream but out-of-frame with pp38 (RB1Bpp38/smGFP). During construction of RB1Bpp38/eGFP, an ORF located downstream of pp38 (LORF12) was partially deleted. In RB1Bpp38/smGFP, however, LORF12 and its immediate 5' upstream sequence was left intact. This report describes the construction, cell culture, and in vivo characterization of RB1Bpp38/eGFP and RB1Bpp38/smGFP. Structural analysis showed that the virus stocks of RB1Bpp38/eGFP and RB1Bpp38/smGFP had incorporated the GFP cassette and were free of contaminating parent virus (RB1B). Moreover, RB1Bpp38/eGFP and RB1Bpp38/smGFP contained two and three and four and five copies of the 132-bp repeats, respectively. Expression analysis showed that the transcription of genes in RB1Bpp38/eGFP-and RB1Bpp38/smGFP-infected chicken embryo fibroblasts (CEFs) were similar to RB1B-infected CEFs, with the notable exception of deletion of a LORF12-specific transcript in RB1Bpp38/ eGFP-infected cells. In CEFs, RB1Bpp38/eGFP and RB1Bpp38/smGFP showed comparable one-step growth kinetics to parental virus (RB1B). RB1Bpp38/eGFP and RB1Bpp38/smGFP, however, showed quite distinct growth characteristics in vivo. Two independent clones of RB1Bpp38/eGFP were highly attenuated, whereas RB1Bpp38/smGFP exhibited pathogenesis similar to parent virus and retained oncogenicity. Our results suggest that the RB1Bpp38/eGFP phenotype could be due to an interference with an in vivo-specific pp38 function via GFP direct fusion, to the deletion of LORF12, or to a targeting of the immune response to eGFP. Because deletion of pp38 was recently found not to fully attenuate very virulent MDV strain MD-5, it is possible that deletion of LORF12 may be at least partially responsible for the attenuation of RB1Bpp38/eGFP. The construction of these viruses and the establishment of cell lines from RB1Bpp38/smGFP provide useful tools for the study of MDV lyric infection in cell culture and in vivo, in studies of the reactivation of MDV from latency, and in the functional analysis of LORF12.