Vaccinia virus (VACV) has been used more extensively for human immunization than any other vaccine. For almost two centuries, VACV was employed to provide cross-protection against variola virus, the causative agent of smallpox, until the disease was eradicated in the late 1970s. Since that time, continued research on VACV has produced a number of modified vaccines with improved safety profiles. Attenuation has been achieved through several strategies, including sequential passage in an alternative host, deletion of specific genes or genetic engineering of viral genes encoding immunomodulatory proteins. Some highly attenuated third-and fourth-generation VACV vaccines are now being considered for stockpiling against a possible re-introduction of smallpox through bioterrorism. Researchers have also taken advantage of the ability of the VACV genome to accommodate additional genetic material to produce novel vaccines against a wide variety of infectious agents, including a recombinant VACV encoding the rabies virus glycoprotein that is administered orally to wild animals. This review provides an in-depth examination of these successive generations of VACV vaccines, focusing on how the understanding of poxviral replication and viral gene function permits the deliberate modification of VACV immunogenicity and virulence.

COVID-19 patients frequently develop neurological symptoms, but the biological underpinnings of these phenomena are unknown. Through single cell RNA-seq and cytokine analyses of CSF and blood from COVID-19 patients with neurological symptoms, we find compartmentalized, CNS specific T cell activation and B cell responses. All COVID-19 cases had CSF anti-SARS-CoV-2 antibodies whose target epitopes diverged from serum antibodies. In an animal model, we find that intrathecal SARS-CoV-2 antibodies are found only during brain infection, and are not elicited by pulmonary infection. We produced CSF-derived monoclonal antibodies from a COVID-19 patient, and find that these mAbs target both anti-viral and anti-neural antigens—including one mAb that reacted to both spike protein and neural tissue. Overall, CSF IgG from 5/7 patients contains anti-neural reactivity. This immune survey reveals evidence of a compartmentalized immune response in the CNS of COVID-19 patients and suggests a role for autoimmunity in neurologic sequelae of COVID-19.

Incorporation of D-amino acids into peptidoglycan is a unique metabolic feature of bacteria. Since D-amino acids are not metabolic substrates in most mammalian tissues, this difference can be exploited to detect living bacteria in vivo. Given the prevalence of D-alanine in peptidoglycan muropeptides, as well as its role in several antibiotic mechanisms, we targeted this amino acid for positron emission tomography (PET) radiotracer development. D-[3-11 C]Alanine and the dipeptide D-[3-11 C]alanyl-Dalanine were synthesized via asymmetric alkylation of glycine-derived Schiff-base precursors with [ 11 C]methyl iodide in the presence of a cinchonidinium phase-transfer catalyst. In cell experiments, both tracers showed accumulation by a wide variety of both Grampositive and Gram-negative pathogens including Staphylococcus aureus and Pseudomonas aeruginosa. In a mouse model of acute bacterial myositis, D-[3-11 C]alanine was accumulated by living microorganisms but was not taken up in areas of sterile inflammation.When compared to existing clinical nuclear imaging tools, specifically 2-deoxy-2-[ 18 F]fluoro-D-glucose and a gallium citrate radiotracer, D-alanine showed more bacteria-specific uptake. Decreased D-[3-11 C]alanine uptake was also observed in antibioticsensitive microbes after antimicrobial therapy, when compared to that in resistant organisms. Finally, prominent uptake of D-[3-11 C]alanine uptake was seen in rodent models of discitis-osteomyelitis and P. aeruginosa pneumonia. These data provide strong justification for clinical translation of D-[3-11 C]alanine to address a number of important human infections.

While as yet there is no vaccine against HIV/AIDS, the results of the phase III Thai trial (RV144) have been encouraging and suggest that further improvements of the prime/boost vaccine combination of a poxvirus and protein are needed. With this aim, in this investigation we have generated derivatives of the candidate vaccinia virus vaccine vector NYVAC with potentially improved functions. This has been achieved by the re-incorporation into the virus genome of two host range genes, K1L and C7L, in conjunction with the removal of the immunomodulatory viral molecule B19, an antagonist of type I interferon action. These novel virus vectors, referred to as NYVAC-C-KC and NYVAC-C-KC-ΔB19R, have acquired relevant biological characteristics, giving higher levels of antigen expression in infected cells, replication-competency in human keratinocytes and dermal fibroblasts, activation of selective host cell signal transduction pathways, and limited virus spread in tissues. Importantly, these replication-competent viruses have been demonstrated to maintain a highly attenuated phenotype.

Compelling evidence points to immune cell infiltration as a critical component of successful immunotherapy. However, there are currently no clinically available, non-invasive methods capable of evaluating immune contexture prior to or during immunotherapy. In this study, we evaluate a T cell-specific PET agent, [18F]F-AraG, as an imaging biomarker predictive of response to checkpoint inhibitor therapy. We determined the specificity of the tracer for activated T cells in vitro and in a virally induced model of rhabdomyosarcoma. Of all immune cells tested, activated human CD8+ effector cells showed the highest accumulation of [18F]F-AraG. Isolation of lymphocytes from the rhabdomyosarcoma tumors showed that more than 80% of the intratumoral signal came from accumulation of [18F]F-AraG in immune cells, primarily CD8+ and CD4+. Longitudinal monitoring of MC38 tumor bearing mice undergoing anti-PD-1 treatment revealed differences in signal between PD-1 and isotype antibody-treated mice early into treatment. The differences in [18F]F-AraG signal were also apparent between responders and non-responders to anti-PD-1 therapy. Importantly, we found that the signal in the tumor draining lymph nodes provides key information about response to anti-PD-1 therapy. Overall, [18F]F-AraG has potential to serve as a much needed immunomonitoring clinical tool for timely evaluation of immunotherapy.