Serological responses to smallpox A33 antigen and monkeypox A35 antigen in healthy people and people living with HIV-1 from Guangzhou, China.

People are expected to have been previously vaccinated with a Vaccinia-based vaccine, as until 1980 smallpox vaccination was a standard protocol in China. It is unclear whether people with smallpox vaccine still have antibody against vaccinia virus (VACV) and cross-antibody against monkeypox virus (MPXV). Herein, we assessed the binding antibodies with antigen of VACV-A33 and MPXV-A35 in the general population and HIV-1 infected patients. Firstly, we detected VACV antibody with A33 protein to evaluate the efficiency of smallpox vaccination. The result show that 29% (23 of 79) of hospital staff (age ≥ 42 years) and 63% (60 of 95) of HIV-positive patients (age ≥ 42 years) from Guangzhou Eighth People's Hospital were able to bind A33. However, among the subjects below 42 years of age, 1.5% (3/198) of the hospital volunteer samples and 1% (1/104) of the samples from HIV patients were positive for antibodies against A33 antigen. Then, we assessed the specific cross-reactive antibodies against MPXV A35 protein. 24% (19 of 79) hospital staff (age〉 = 42 years) and 44% (42 of 95) of HIV-positive patients (age〉 = 42 years) were positive. 98% (194/198) of the hospital staff and 99% (103/104) of the HIV patients had no A35-binding antibodies. Further, we found significant sex differences for the reactivity to A35 antigen were observed in HIV population, but no significant sex differences in hospital staff. Further, we analyzed the positivity rate of anti-A35 antibody of men who have sex with men (MSM) and non-MSM in HIV patients (age〉 = 42years). We found that 47% of no-MSM population and 40% of MSM population were positive for A35 antigen, with no significant difference. Lastly, we found only 59 samples were positive for anti-A33 IgG and anti-A35 IgG in all participants. Together, we demonstrated A33 and A35 antigens binding antibodies were detected in HIV patients and general population who were older than 42 years, and cohort studies only provided data of serological detection to support early response to monkeypox outbreak.

Estimation of anti-orthopoxvirus immunity in Moscow residents and potential risks of spreading Monkeypox virus.

WHO has declared the outbreak of monkeypox as a public health emergency of international concern. In less than three months, monkeypox was detected in more than 30 000 people and spread to more than 80 countries around the world. It is believed that the immunity formed to smallpox vaccine can protect from monkeypox infection with high efficiency. The widespread use of Vaccinia virus has not been carried out since the 1980s, which raises the question of the level of residual immunity among the population and the identification of groups requiring priority vaccination. We conducted a cross-sectional serological study of remaining immunity among Moscow residents. To do this, a collection of blood serum samples of age group over 30 years old was formed, an in-house ELISA test system was developed, and a virus neutralization protocol was set up. Serum samples were examined for the presence of IgG antibodies against Vaccinia virus (n=2908), as well as for the ability to neutralize plaque formation with a Vaccinia virus MNIIVP-10 strain (n=299). The results indicate the presence of neutralizing antibody titer of 1/20 or more in 33.3 to 53.2% of people older than 45 years. Among people 30-45 years old who probably have not been vaccinated, the proportion with virus neutralizing antibodies ranged from 3.2 to 6.7%. Despite the higher level of antibodies in age group older than 66 years, the proportion of positive samples in this group was slightly lower than in people aged 46-65 years. The results indicate the priority of vaccination in groups younger than 45, and possibly older than 66 years to ensure the protection of the population in case of spread of monkeypox among Moscow residents. The herd immunity level needed to stop the circulation of the virus should be at least 50.25 - 65.28%.

CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX.

Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assisted-recombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies.

Imaging of Virus-Infected Cells with Soft X-ray Tomography.

Viruses are obligate parasites that depend on a host cell for replication and survival. Consequently, to fully understand the viral processes involved in infection and replication, it is fundamental to study them in the cellular context. Often, viral infections induce significant changes in the subcellular organization of the host cell due to the formation of viral factories, alteration of cell cytoskeleton and/or budding of newly formed particles. Accurate 3D mapping of organelle reorganization in infected cells can thus provide valuable information for both basic virus research and antiviral drug development. Among the available techniques for 3D cell imaging, cryo-soft X-ray tomography stands out for its large depth of view (allowing for 10 µm thick biological samples to be imaged without further thinning), its resolution (about 50 nm for tomographies, sufficient to detect viral particles), the minimal requirements for sample manipulation (can be used on frozen, unfixed and unstained whole cells) and the potential to be combined with other techniques (i.e., correlative fluorescence microscopy). In this review we describe the fundamentals of cryo-soft X-ray tomography, its sample requirements, its advantages and its limitations. To highlight the potential of this technique, examples of virus research performed at BL09-MISTRAL beamline in ALBA synchrotron are also presented.

Vaccinia Virus Activation and Antagonism of Cytosolic DNA Sensing.

Cells express multiple molecules aimed at detecting incoming virus and infection. Recognition of virus infection leads to the production of cytokines, chemokines and restriction factors that limit virus replication and activate an adaptive immune response offering long-term protection. Recognition of cytosolic DNA has become a central immune sensing mechanism involved in infection, autoinflammation, and cancer immunotherapy. Vaccinia virus (VACV) is the prototypic member of the family Poxviridae and the vaccine used to eradicate smallpox. VACV harbors enormous potential as a vaccine vector and several attenuated strains are currently being developed against infectious diseases. In addition, VACV has emerged as a popular oncolytic agent due to its cytotoxic capacity even in hypoxic environments. As a poxvirus, VACV is an unusual virus that replicates its large DNA genome exclusively in the cytoplasm of infected cells. Despite producing large amounts of cytosolic DNA, VACV efficiently suppresses the subsequent innate immune response by deploying an arsenal of proteins with capacity to disable host antiviral signaling, some of which specifically target cytosolic DNA sensing pathways. Some of these strategies are conserved amongst orthopoxviruses, whereas others are seemingly unique to VACV. In this review we provide an overview of the VACV replicative cycle and discuss the recent advances on our understanding of how VACV induces and antagonizes innate immune activation via cytosolic DNA sensing pathways. The implications of these findings in the rational design of vaccines and oncolytics based on VACV are also discussed.

EPPIC (Efficient Purification by Parental Inducer Constraint) Platform for Rapid Generation of Recombinant Vaccinia Viruses.

Vaccinia virus (VACV) was successfully used as a vaccine in the smallpox eradication campaign. Since then, it has been widely used in the development of vaccine and therapeutic vectors. However, methods of generating and purifying recombinant VACVs (rVACVs) are often time-consuming, cumbersome, and in some cases require specialized cell lines or equipment. Here, we describe a novel EPPIC (Efficient Purification by Parental Inducer Constraint) platform for the rapid generation of rVACVs using a replication-inducible VACV (vIND) as a parental virus for homologous recombination. Purification of the rVACV from the parental vIND is achieved by two serial passages in the absence of inducer (i.e., parental inducer "constraint") in standard laboratory cell lines, without the need for specialized equipment, within 1 week. We determined the optimal conditions for homologous recombination and serial purification and generated a suite of vIND parental viruses to facilitate customization of the platform. Importantly, the EPPIC platform can be adapted to rapidly generate replication-deficient and replication-competent rVACVs expressing vaccine or therapeutic antigens, with or without screening markers, by simple modifications to a DNA shuttle vector, thus allowing the rapid development, updating, and refinement of personalized or custom vaccines and therapeutic vectors in a matter of days.

Poxviruses as Gene Therapy Vectors: Generating Poxviral Vectors Expressing Therapeutic Transgenes.

Treatments with poxvirus vectors can have long-lasting immunological impact in the host, and thus they have been extensively studied to treat diseases and for vaccine development. More importantly, the oncolytic properties of poxviruses have led to their development as cancer therapeutics. Two poxviruses, vaccinia virus (VACV) and myxoma virus (MYXV), have been extensively studied as virotherapeutics with promising results. Vaccinia virus vectors have advanced to the clinic and have been tested as oncolytic therapeutics for several cancer types with successes in phase I/II clinical trials. In addition to oncolytic applications, MYXV has been explored for additional applications including immunotherapeutics, purging of cancer progenitor cells, and treatments for graft-versus-host diseases. These novel therapeutic applications have encouraged its advancement into clinical trials. To meet the demands of different treatment needs, VACV and MYXV can be genetically engineered to express therapeutic transgenes. The engineering process used in poxvirus vectors can be very different from that of other DNA virus vectors (e.g., the herpesviruses). This chapter is intended to serve as a guide to those wishing to engineer poxvirus vectors for therapeutic transgene expression and to produce viral preparations for preclinical studies.