Combination of Extended Antivirals With Antiretrovirals for Severe Mpox in Advanced Human Immunodeficiency Virus Infection: Case Series of 4 Patients.

To gauge the safety and utility of extended tecovirimat/cidofovir for severe mpox, here we report our experience caring for 4 patients with mpox and advanced human immunodeficiency virus (HIV) at the Hospitals of the University of Pennsylvania during the 2022 global outbreak. Three patients had recurrent courses complicated by superinfections, coinfections and insufficient nutrition/housing, requiring extended tecovirimat (5-16 weeks) and cidofovir (1-12 doses) with probenecid and fluids. At follow-up, patients had undetectable HIV RNA on antiretrovirals, improved ulcers and stable renal function on antivirals. Serology guided cessation for one 7-month cidofovir course. Overall findings support a comprehensive approach of prolonged tecovirimat/cidofovir with antiretrovirals for severe mpox, while addressing social factors.

Mpox: Keep it on the differential.

In its current global outbreak, mpox has exhibited several novel clinical presentations that clinicians should be aware of so they can recognize it if they see it. Although the case rate has decreased, mpox could linger at a low rate or resurface in other populations and thus should remain in the differential diagnosis in patients presenting with potential infections after intimate encounters.

Early Outcomes of SARS-CoV-2 Infection in a Multisite Prospective Cohort of Inpatient Veterans.

Background: Over 870 000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have occurred among Veterans Health Administration users, and 24 000 have resulted in death. We examined early outcomes of SARS-CoV-2 infection in hospitalized veterans. Methods: In an ongoing, prospective cohort study, we enrolled veterans age ≥18 tested for SARS-CoV-2 and hospitalized at 15 Department of Veterans Affairs medical centers between February 2021 and June 2022. We estimated adjusted odds ratios (aORs), adjusted incidence rate ratios (aIRRs), and adjusted hazard ratios (aHRs) for maximum illness severity within 30 days of study entry (defined using the 4-category VA Severity Index for coronavirus disease 2019 [COVID-19]), as well as length of hospitalization and rehospitalization within 60 days, in relationship with demographic characteristics, Charlson comorbidity index (CCI), COVID-19 vaccination, and calendar period of enrollment. Results: The 542 participants included 329 (61%) who completed a primary vaccine series (with or without booster; "vaccinated"), 292 (54%) enrolled as SARS-CoV-2-positive, and 503 (93%) men, with a mean age of 64.4 years. High CCI scores (≥5) occurred in 61 (44%) vaccinated and 29 (19%) unvaccinated SARS-CoV-2-positive participants. Severe illness or death occurred in 29 (21%; 6% died) vaccinated and 31 (20%; 2% died) unvaccinated SARS-CoV-2-positive participants. SARS-CoV-2-positive inpatients per unit increase in CCI had greater multivariable-adjusted odds of severe illness (aOR, 1.21; 95% CI, 1.01-1.45), more hospitalization days (aIRR, 1.06; 95% CI, 1.03-1.10), and rehospitalization (aHR, 1.07; 95% CI, 1.01-1.12). Conclusions: In a cohort of hospitalized US veterans with SARS-CoV-2 infection, those with a higher CCI had more severe COVID-19 illness, more hospital days, and rehospitalization, after adjusting for vaccination status, age, sex, and calendar period.

ICTV Virus Taxonomy Profile: Poxviridae 2023.

Poxviridae is a family of enveloped, brick-shaped or ovoid viruses. The genome is a linear molecule of dsDNA (128-375 kbp) with covalently closed ends. The family includes the sub-families Entomopoxvirinae, whose members have been found in four orders of insects, and Chordopoxvirinae, whose members are found in mammals, birds, reptiles and fish. Poxviruses are important pathogens in various animals, including humans, and typically result in the formation of lesions, skin nodules, or disseminated rash. Infections can be fatal. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Poxviridae, which is available at ictv.global/report/poxviridae.

Monkeypox (Mpox) requires continued surveillance, vaccines, therapeutics and mitigating strategies.

The widespread outbreak of the monkeypox virus (MPXV) recognized in 2022 poses new challenges for public healthcare systems worldwide. With more than 86,000 people infected, there is concern that MPXV may become endemic outside of its original geographical area leading to repeated human spillover infections or continue to be spread person-to-person. Fortunately, classical public health measures (e.g., isolation, contact tracing and quarantine) and vaccination have blunted the spread of the virus, but cases are continuing to be reported in 28 countries in March 2023. We describe here the vaccines and drugs available for the prevention and treatment of MPXV infections. However, although their efficacy against monkeypox (mpox) has been established in animal models, little is known about their efficacy in the current outbreak setting. The continuing opportunity for transmission raises concerns about the potential for evolution of the virus and for expansion beyond the current risk groups. The priorities for action are clear: 1) more data on the efficacy of vaccines and drugs in infected humans must be gathered; 2) global collaborations are necessary to ensure that government authorities work with the private sector in developed and low and middle income countries (LMICs) to provide the availability of treatments and vaccines, especially in historically endemic/enzootic areas; 3) diagnostic and surveillance capacity must be increased to identify areas and populations where the virus is present and may seed resurgence; 4) those at high risk of severe outcomes (e.g., immunocompromised, untreated HIV, pregnant women, and inflammatory skin conditions) must be informed of the risk of infection and be protected from community transmission of MPXV; 5) engagement with the hardest hit communities in a non-stigmatizing way is needed to increase the understanding and acceptance of public health measures; and 6) repositories of monkeypox clinical samples, including blood, fluids, tissues and lesion material must be established for researchers. This MPXV outbreak is a warning that pandemic preparedness plans need additional coordination and resources. We must prepare for continuing transmission, resurgence, and repeated spillovers of MPXV.

A small molecule that targets the processivity factor of molluscum contagiosum virus has therapeutic potential.

Molluscum contagiosum (MC) is an infectious disease that occurs only in humans with a tropism that is narrowly restricted to the outermost epidermal layer of the skin. Molluscum contagiosum virus (MCV) is the causative agent of MC which produces skin lesions that can persist for months to several years. MCV is efficiently transmitted by direct physical contact or by indirect contact with fomites. MC is most prevalent in children and immune compromised patients. The failure to develop a drug that targets MCV replication has been hampered for decades by the inability to propagate MCV in cell culture. To address this dilemma, we recently engineered a surrogate poxvirus expressing the MCV processivity factor (mD4) as the drug target. The mD4 protein is essential for viral replication by keeping the viral polymerase tethered to the DNA template. In this study we have designed and synthesized a lead compound (7269) that is able to prevent mD4 dependent processive DNA synthesis in vitro (IC50 = 6.8 µM) and effectively inhibit propagation of the mD4-VV surrogate virus in BSC-1 cells (EC50 = 13.2 µM) with negligible cytotoxicity. In human liver microsomes, 7269 was shown to be stable for almost 2 h. When tested for penetration into human cadaver skin in a formulated gel, the level of 7269 in the epidermal layer was nearly 100 times the concentration (EC50) needed to inhibit propagation of the mD4-VV surrogate virus in BSC-1 cells. The gel formulated 7269 was scored as a non-irritant on skin and shown to have a shelf-life that was completely stable after several months. In summary, 7269 is a potential Lead for becoming the first MCV anti-viral compound to treat MC and thereby, addresses this unmet medical need that has persisted for many decades.

Mpox Evolution: Has the Current Outbreak Revealed a Pox on "U"?

The recent mpox (monkeypox) outbreak has prompted genomic studies to track global spread of the disease. These studies have also revealed unexpected patterns of mutations that implicate the action of the immune defense APOBEC3 family of enzymes, which catalyze conversion of cytosine (C) to uracil (U) in DNA, in viral evolution. As poxviruses have conventionally been regarded as slow-evolving viruses, the rapid emergence of APOBEC3 mutational signatures begs a series of important and open questions regarding how host-pathogen interactions may have changed and whether these mutations are bystanders or have roles in pathogenesis.

Use of JYNNEOS (Smallpox and Monkeypox Vaccine, Live, Nonreplicating) for Preexposure Vaccination of Persons at Risk for Occupational Exposure to Orthopoxviruses: Recommendations of the Advisory Committee on Immunization Practices - United States, 2022.

Certain laboratorians and health care personnel can be exposed to orthopoxviruses through occupational activities. Because orthopoxvirus infections resulting from occupational exposures can be serious, the Advisory Committee on Immunization Practices (ACIP) has continued to recommend preexposure vaccination for these persons since 1980 (1), when smallpox was eradicated (2). In 2015, ACIP made recommendations for the use of ACAM2000, the only orthopoxvirus vaccine available in the United States at that time (3). During 2020-2021, ACIP considered evidence for use of JYNNEOS, a replication-deficient Vaccinia virus vaccine, as an alternative to ACAM2000. In November 2021, ACIP unanimously voted in favor of JYNNEOS as an alternative to ACAM2000 for primary vaccination and booster doses. With these recommendations for use of JYNNEOS, two vaccines (ACAM2000 and JYNNEOS) are now available and recommended for preexposure prophylaxis against orthopoxvirus infection among persons at risk for such exposures.

Short-term and longer-term protective immune responses generated by subunit vaccination with smallpox A33, B5, L1 or A27 proteins adjuvanted with aluminum hydroxide and CpG in mice challenged with vaccinia virus.

Smallpox, a contagious and deadly disease caused by variola virus, was eradicated by a strategy that included vaccination with vaccinia virus, a live-virus vaccine. Because the threat of bioterrorism with smallpox persists and infections with zoonotic poxvirus infections like monkeypox continue, and there may be a time when an alternative vaccine platform is needed, recombinant-subunit vaccine strategies for poxviruses have been pursued. Our prior work focused on understanding the immune responses generated to vaccine-formulations containing the virus protein L1. In this work, we examine vaccine-formulations with additional key protein targets: A33 and B5 (components of the extracellular virus) and another protein on the mature virus (A27) adjuvanted with aluminum hydroxide (AH) with and without CpG- oligonucleotide. Each vaccine was formulated to allow either adsorption or non-adsorption of the protein (and CpG) to AH. Mice given a prime and single boost produced long-lasting antibody responses. A second boost (given ~5-months after the first) further increased antibody titers. Similar to our prior findings with L1 vaccine-formulations, the most protective A33 vaccine-formulations included CpG, resulted in the generation of IgG2a-antibody responses. Unlike the prior findings with L1 (where formulations that adsorbed both the protein and the CpG to AH resulted in 100% survival after challenge and minimal weight loss), the AH-adsorption status of A33 and CpG did not play as important a role, since both AH-adsorbed and non-adsorbed groups lost weight after challenge and had similar survival. Vaccination with B5-formulations gave different results. While CpG-containing formulations were the only ones that generated IgG2a-antibody responses, the vaccine-formulation that adsorbed B5 to AH (without CpG) was as equally effective in protecting mice after challenge. These results indicate that the mechanism of how antibodies against A33 and B5 protect differ. The data also show the complexity of designing optimized vaccine-formulations containing multiple adjuvants and recombinant protein-based antigens.

Working Safely with Vaccinia Virus: Laboratory Technique and Review of Published Cases of Accidental Laboratory Infections with Poxviruses.

Vaccinia virus, the prototype Orthopoxvirus, is widely used in the laboratory as a model system to study various aspects of viral biology and virus-host interactions, as a protein expression system, as a vaccine vector, and as an oncolytic agent. The ubiquitous use of vaccinia viruses in laboratories around the world raises certain safety concerns because the virus can be a pathogen in individuals with immunological and dermatological abnormalities, and on occasion can cause serious problems in normal hosts. This chapter reviews standard operating procedures when working with vaccinia virus and reviews published cases of accidental laboratory infections with poxviruses.

Mutation and structure guided discovery of an antiviral small molecule that mimics an essential C-Terminal tripeptide of the vaccinia D4 processivity factor.

The smallpox virus (variola) remains a bioterrorism threat since a majority of the human population has never been vaccinated. In the event of an outbreak, at least two drugs against different targets of variola are critical to circumvent potential viral mutants that acquire resistance. Vaccinia virus (VACV) is the model virus used in the laboratory for studying smallpox. The VACV processivity factor D4 is an ideal therapeutic target since it is both essential and specific for poxvirus replication. Recently, we identified a tripeptide (Gly-Phe-Ile) motif at the C-terminus of D4 that is conserved among poxviruses and is necessary for maintaining protein function. In the current work, a virtual screening for small molecule mimics of the tripeptide identified a thiophene lead that effectively inhibited VACV, cowpox virus, and rabbitpox virus in cell culture (EC50 = 8.4-19.7 µM) and blocked in vitro processive DNA synthesis (IC50 = 13.4 µM). Compound-binding to D4 was demonstrated through various biophysical methods and a dose-dependent retardation of the proteolysis of D4 proteins. This study highlights an inhibitor design strategy that exploits a susceptible region of the protein and identifies a novel scaffold for a broad-spectrum poxvirus inhibitor.

Identifying Host Factors Associated with DNA Replicated During Virus Infection.

Viral DNA genomes replicating in cells encounter a myriad of host factors that facilitate or hinder viral replication. Viral proteins expressed early during infection modulate host factors interacting with viral genomes, recruiting proteins to promote viral replication, and limiting access to antiviral repressors. Although some host factors manipulated by viruses have been identified, we have limited knowledge of pathways exploited during infection and how these differ between viruses. To identify cellular processes manipulated during viral replication, we defined proteomes associated with viral genomes during infection with adenovirus, herpes simplex virus and vaccinia virus. We compared enrichment of host factors between virus proteomes and confirmed association with viral genomes and replication compartments. Using adenovirus as an illustrative example, we uncovered host factors deactivated by early viral proteins, and identified a subgroup of nucleolar proteins that aid virus replication. Our data sets provide valuable resources of virus-host interactions that affect proteins on viral genomes.

A novel target and approach for identifying antivirals against molluscum contagiosum virus.

The dermatological disease molluscum contagiosum (MC) presents as lesions restricted solely to the skin. The poxvirus molluscum contagiosum virus (MCV) is responsible for this skin disease that is easily transmitted through casual contact among all populations, with greater frequency in children and immunosuppressed individuals. In addition, sexual transmission of MCV in adolescents and adults is a health concern. Although the skin lesions ultimately resolve in immunocompetent individuals, they can persist for extended periods, be painful, and result in scarring. Treatment is problematic, and there is no drug that specifically targets MCV. The inability of MCV to propagate in cell culture has impeded drug development. To overcome these barriers, we integrated three new developments. First, we identified a new MCV drug target (mD4) that is essential for processive DNA synthesis in vitro. Second, we discovered a small chemical compound that binds to mD4 and prevents DNA synthesis in vitro. Third, and most significant, we engineered a hybrid vaccinia virus (mD4-VV) in which the natural vaccinia D4 (vD4) gene is replaced by the mD4 target gene. This hybrid virus is dependent on mD4 for viral growth in culture and is inhibited by the small compound. This target system provides, for the first time, a platform and approach for the discovery and evaluation of new therapeutics that can be used to treat MC.

Adsorption of recombinant poxvirus L1-protein to aluminum hydroxide/CpG vaccine adjuvants enhances immune responses and protection of mice from vaccinia virus challenge.

The stockpiling of live vaccinia virus vaccines has enhanced biopreparedness against the intentional or accidental release of smallpox. Ongoing research on future generation smallpox vaccines is providing key insights into protective immune responses as well as important information about subunit-vaccine design strategies. For protein-based recombinant subunit vaccines, the formulation and stability of candidate antigens with different adjuvants are important factors to consider for vaccine design. In this work, a non-tagged secreted L1-protein, a target antigen on mature virus, was expressed using recombinant baculovirus technology and purified. To identify optimal formulation conditions for L1, a series of biophysical studies was performed over a range of pH and temperature conditions. The overall physical stability profile was summarized in an empirical phase diagram. Another critical question to address for development of an adjuvanted vaccine was if immunogenicity and protection could be affected by the interactions and binding of L1 to aluminum salts (Alhydrogel) with and without a second adjuvant, CpG. We thus designed a series of vaccine formulations with different binding interactions between the L1 and the two adjuvants, and then performed a series of vaccination-challenge experiments in mice including measurement of antibody responses and post-challenge weight loss and survival. We found that better humoral responses and protection were conferred with vaccine formulations when the L1-protein was adsorbed to Alhydrogel. These data demonstrate that designing vaccine formulation conditions to maximize antigen-adjuvant interactions is a key factor in smallpox subunit-vaccine immunogenicity and protection.

Working safely with vaccinia virus: laboratory technique and review of published cases of accidental laboratory infections.

Vaccinia virus (VACV), the prototype orthopoxvirus, is widely used in the laboratory as a model system to study various aspects of viral biology and virus-host interactions, as a protein expression system, as a vaccine vector, and as an oncolytic agent. The ubiquitous use of VACVs in the laboratory raises certain safety concerns because the virus can be a pathogen in individuals with immunological and dermatological abnormalities, and on occasion can cause serious problems in normal hosts. This chapter reviews standard operating procedures when working with VACV and reviews published cases on accidental laboratory infections.

Enzyme-linked immunosorbent assay (ELISA) and blocking with bovine serum albumin (BSA)--not all BSAs are alike.

The enzyme-linked immunosorbent assay (ELISA) is an extremely common and powerful laboratory technique for detecting proteins by antibodies. Researchers frequently use bovine serum albumin (BSA) as a blocking agent to prevent non-specific binding of antigens and antibodies to the microtiter well. While studying the interactions of the vaccinia virus complement control protein (VCP) with complement, we found non-specific binding of VCP to BSA and identify a BSA preparation that did not result in non-specific binding. This work draws attention to the fact that not all BSA preparations are alike. It also highlights the need to perform critical controls to ensure that ELISA reactants do not inappropriately bind to the blocking agent.

Elucidating the role of the complement control protein in monkeypox pathogenicity.

Monkeypox virus (MPXV) causes a smallpox-like disease in humans. Clinical and epidemiological studies provide evidence of pathogenicity differences between two geographically distinct monkeypox virus clades: the West African and Congo Basin. Genomic analysis of strains from both clades identified a ∼10 kbp deletion in the less virulent West African isolates sequenced to date. One absent open reading frame encodes the monkeypox virus homologue of the complement control protein (CCP). This modulatory protein prevents the initiation of both the classical and alternative pathways of complement activation. In monkeypox virus, CCP, also known as MOPICE, is a ∼24 kDa secretory protein with sequence homology to this superfamily of proteins. Here we investigate CCP expression and its role in monkeypox virulence and pathogenesis. CCP was incorporated into the West African strain and removed from the Congo Basin strain by homologous recombination. CCP expression phenotypes were confirmed for both wild type and recombinant monkeypox viruses and CCP activity was confirmed using a C4b binding assay. To characterize the disease, prairie dogs were intranasally infected and disease progression was monitored for 30 days. Removal of CCP from the Congo Basin strain reduced monkeypox disease morbidity and mortality, but did not significantly decrease viral load. The inclusion of CCP in the West African strain produced changes in disease manifestation, but had no apparent effect on disease-associated mortality. This study identifies CCP as an important immuno-modulatory protein in monkeypox pathogenesis but not solely responsible for the increased virulence seen within the Congo Basin clade of monkeypox virus.

Treating tumors with a vaccinia virus expressing IFNß illustrates the complex relationships between oncolytic ability and immunogenicity.

Since previous work using a nonreplicating adenovirus-expressing mouse interferon-ß (Ad.mIFNß) showed promising preclinical activity, we postulated that a vector-expressing IFNß at high levels that could also replicate would be even more beneficial. Accordingly a replication competent, recombinant vaccinia viral vector-expressing mIFNß (VV.mIFNß) was tested. VV.mIFNß-induced antitumor responses in two syngeneic mouse flank models of lung cancer. Although VV.mIFNß had equivalent in vivo efficacy in both murine tumor models, the mechanisms of tumor killing were completely different. In LKRM2 tumors, viral replication was minimal and the tumor killing mechanism was due to activation of immune responses through induction of a local inflammatory response and production of antitumor CD8 T-cells. In contrast, in TC-1 tumors, the vector replicated well, induced an innate immune response, but antitumor activity was primarily due to a direct oncolytic effect. However, the VV.mIFNß vector was able to augment the efficacy of an antitumor vaccine in the TC-1 tumor model in association with increased numbers of infiltrating CD8 T-cells. These data show the complex relationships between oncolytic viruses and the immune system which, if understood and harnessed correctly, could potentially be used to enhance the efficacy of immunotherapy.