Adaptive Immune Response to Vaccinia Virus LIVP Infection of BALB/c Mice and Protection against Lethal Reinfection with Cowpox Virus.

Mass vaccination has played a critical role in the global eradication of smallpox. Various vaccinia virus (VACV) strains, whose origin has not been clearly documented in most cases, have been used as live vaccines in different countries. These VACV strains differed in pathogenicity towards various laboratory animals and in reactogenicity exhibited upon vaccination of humans. In this work, we studied the development of humoral and cellular immune responses in BALB/c mice inoculated intranasally (i.n.) or intradermally (i.d.) with the VACV LIVP strain at a dose of 105 PFU/mouse, which was used in Russia as the first generation smallpox vaccine. Active synthesis of VACV-specific IgM in the mice occurred on day 7 after inoculation, reached a maximum on day 14, and decreased by day 29. Synthesis of virus-specific IgG was detected only from day 14, and the level increased significantly by day 29 after infection of the mice. Immunization (i.n.) resulted in significantly higher production of VACV-specific antibodies compared to that upon i.d. inoculation of LIVP. There were no significant differences in the levels of the T cell response in mice after i.n. or i.d. VACV administration at any time point. The maximum level of VACV-specific T-cells was detected on day 14. By day 29 of the experiment, the level of VACV-specific T-lymphocytes in the spleen of mice significantly decreased for both immunization procedures. On day 30 after immunization with LIVP, mice were infected with the cowpox virus at a dose of 46 LD50. The i.n. immunized mice were resistant to this infection, while 33% of i.d. immunized mice died. Our findings indicate that the level of the humoral immune response to vaccination may play a decisive role in protection of animals from orthopoxvirus reinfection.

A class of viral inducer of degradation of the necroptosis adaptor RIPK3 regulates virus-induced inflammation.

The vaccine strain against smallpox, vaccinia virus (VACV), is highly immunogenic yet causes relatively benign disease. These attributes are believed to be caused by gene loss in VACV. Using a targeted small interfering RNA (siRNA) screen, we identified a viral inhibitor found in cowpox virus (CPXV) and other orthopoxviruses that bound to the host SKP1-Cullin1-F-box (SCF) machinery and the essential necroptosis kinase receptor interacting protein kinase 3 (RIPK3). This "viral inducer of RIPK3 degradation" (vIRD) triggered ubiquitination and proteasome-mediated degradation of RIPK3 and inhibited necroptosis. In contrast to orthopoxviruses, the distantly related leporipoxvirus myxoma virus (MYXV), which infects RIPK3-deficient hosts, lacks a functional vIRD. Introduction of vIRD into VACV, which encodes a truncated and defective vIRD, enhanced viral replication in mice. Deletion of vIRD reduced CPXV-induced inflammation, viral replication, and mortality, which were reversed in RIPK3- and MLKL-deficient mice. Hence, vIRD-RIPK3 drives pathogen-host evolution and regulates virus-induced inflammation and pathogenesis.

Cowpox virus encodes a protein that binds B7.1 and B7.2 and subverts T cell costimulation.

Costimulation is required for optimal T cell activation, yet it is unclear whether poxviruses dedicatedly subvert costimulation during infection. Here, we report that the secreted M2 protein encoded by cowpox virus (CPXV) specifically interacts with human and murine B7.1 (CD80) and B7.2 (CD86). We also show that M2 competes with CD28 and CTLA4 for binding to cell surface B7 ligands, with stronger efficacy against CD28. Functionally, recombinant M2 and culture supernatants from wild-type (WT) but not M2-deficient (∆M2) CPXV-infected cells can potently suppress B7 ligand-mediated T cell proliferation and interleukin-2 (IL-2) production. Furthermore, we observed increased antiviral CD4 and CD8 T cell responses in C57BL/6 mice challenged by ∆M2 CPXV compared with WT virus. These differences in immune responses to ∆M2 and WT CPXV were not observed in CD28-deficient mice. Taken together, our findings define a mechanism of viral sabotage of T cell activation that highlights the role of CD28 costimulation in host defense against poxvirus infections.

Atypical Cowpox Virus Infection in Smallpox-Vaccinated Patient, France.

We report a case of atypical cowpox virus infection in France in 2016. The patient sought care for thoracic lesions after injury from the sharp end of a metallic guardrail previously stored in the ground. We isolated a cowpox virus from the lesions and sequenced its whole genome. The patient reported that he had been previously vaccinated against smallpox. We describe an alternative route of cowpox virus infection and raise questions about the immunological status of smallpox-vaccinated patients for circulating orthopoxviruses.

Epidemiological Investigations of Four Cowpox Virus Outbreaks in Alpaca Herds, Germany.

Four cowpox virus (CPXV) outbreaks occurred in unrelated alpaca herds in Eastern Germany during 2012-2017. All incidents were initially noticed due to severe, generalized, and finally lethal CPXV infections, which were confirmed by testing of tissue and serum samples. As CPXV-infection has been described in South American camelids (SACs) only three times, all four herds were investigated to gain a deeper understanding of CPXV epidemiology in alpacas. The different herds were investigated twice, and various samples (serum, swab samples, and crusts of suspicious pox lesions, feces) were taken to identify additionally infected animals. Serum was used to detect CPXV-specific antibodies by performing an indirect immunofluorescence assay (iIFA); swab samples, crusts, and feces were used for detection of CPXV-specific DNA in a real-time PCR. In total, 28 out of 107 animals could be identified as affected by CPXV, by iIFA and/or PCR. Herd seroprevalence ranged from 16.1% to 81.2%. To investigate the potential source of infection, wild small mammals were trapped around all alpaca herds. In two herds, CPXV-specific antibodies were found in the local rodent population. In the third herd, CPXV could be isolated from a common vole (Microtus arvalis) found drowned in a water bucket used to water the alpacas. Full genome sequencing and comparison with the genome of a CPXV from an alpaca from the same herd reveal 99.997% identity, providing further evidence that the common vole is a reservoir host and infection source of CPXV. Only in the remaining fourth herd, none of the trapped rodents were found to be CPXV-infected. Rodents, as ubiquitous reservoir hosts, in combination with increasingly popular alpacas, as susceptible species, suggest an enhanced risk of future zoonotic infections.

Seasonal recurrence of cowpox virus outbreaks in captive cheetahs (Acinonyx jubatus).

Cowpox virus infections in captive cheetahs (Acinonyx jubatus) with high morbidity and mortality have already been reported in the UK and Russia in the 1970s. However, most of the reported cases have been singular events. Here, we report a total of five cowpox virus outbreaks in cheetahs in the same safari park in Denmark between 2010 and 2014. Nine cheetahs showed varying severity of clinical disease; two of them died (22%). All episodes occurred between August and October of the respective year. No other carnivores kept at the same institution nor the keepers taking care of the animals were clinically affected. The clinical picture of cowpox was confirmed by extensive laboratory investigations including histopathological and molecular analyses as well as cell culture isolation of a cowpox virus. High anti-orthopoxvirus antibody titers were detected in all 9 diseased cheetahs compared to seven contact cheetahs without clinical signs and 13 cheetahs not in direct contact. Additionally, whole genome sequencing from one sample of each cluster with subsequent phylogenetic analysis showed that the viruses from different outbreaks have individual sequences but clearly form a clade distinct from other cowpox viruses. However, the intra-clade distances are still larger than those usually observed within clades of one event. These findings indicate multiple and separate introductions of cowpox virus, probably from wild rodent populations, where the virus keeps circulating naturally and is only sporadically introduced into the cheetahs. Sero-positivity of voles (Arvicola amphibious) caught in zoo grounds strengthens this hypothesis. As a consequence, recommendations are given for medical and physical management of diseased cheetahs, for hygienic measures as well as for pre-shipment isolation before cheetah export from zoo grounds.

Equination (inoculation of horsepox): An early alternative to vaccination (inoculation of cowpox) and the potential role of horsepox virus in the origin of the smallpox vaccine.

For almost 150 years after Edward Jenner had published the "Inquiry" in 1798, it was generally assumed that the cowpox virus was the vaccine against smallpox. It was not until 1939 when it was shown that vaccinia, the smallpox vaccine virus, was serologically related but different from the cowpox virus. In the absence of a known natural host, vaccinia has been considered to be a laboratory virus that may have originated from mutational or recombinational events involving cowpox virus, variola viruses or some unknown ancestral Orthopoxvirus. A favorite candidate for a vaccinia ancestor has been the horsepox virus. Edward Jenner himself suspected that cowpox derived from horsepox and he also believed that "matter" obtained from either disease could be used as preventative of smallpox. During the 19th century, inoculation with cowpox (vaccination) was used in Europe alongside with inoculation with horsepox (equination) to prevent smallpox. Vaccine-manufacturing practices during the 19th century may have resulted in the use of virus mixtures, leading to different genetic modifications that resulted in present-day vaccinia strains. Horsepox, a disease previously reported only in Europe, has been disappearing on that continent since the beginning of the 20th century and now seems to have become extinct, although the virus perhaps remains circulating in an unknown reservoir. Genomic sequencing of a horsepox virus isolated in Mongolia in 1976 indicated that, while closely related to vaccinia, this horsepox virus contained additional, potentially ancestral sequences absent in vaccinia. Recent genetic analyses of extant vaccinia viruses have revealed that some strains contain ancestral horsepox virus genes or are phylogenetically related to horsepox virus. We have recently reported that a commercially produced smallpox vaccine, manufactured in the United States in 1902, is genetically highly similar to horsepox virus, providing a missing link in this 200-year-old mystery.

Cross-Neutralizing and Protective Human Antibody Specificities to Poxvirus Infections.

Monkeypox (MPXV) and cowpox (CPXV) are emerging agents that cause severe human infections on an intermittent basis, and variola virus (VARV) has potential for use as an agent of bioterror. Vaccinia immune globulin (VIG) has been used therapeutically to treat severe orthopoxvirus infections but is in short supply. We generated a large panel of orthopoxvirus-specific human monoclonal antibodies (Abs) from immune subjects to investigate the molecular basis of broadly neutralizing antibody responses for diverse orthopoxviruses. Detailed analysis revealed the principal neutralizing antibody specificities that are cross-reactive for VACV, CPXV, MPXV, and VARV and that are determinants of protection in murine challenge models. Optimal protection following respiratory or systemic infection required a mixture of Abs that targeted several membrane proteins, including proteins on enveloped and mature virion forms of virus. This work reveals orthopoxvirus targets for human Abs that mediate cross-protective immunity and identifies new candidate Ab therapeutic mixtures to replace VIG.

Small particle aerosol inoculation of cowpox Brighton Red in rhesus monkeys results in a severe respiratory disease.

Cowpox virus (CPXV) inoculation of nonhuman primates (NHPs) has been suggested as an alternate model for smallpox (Kramski et al., 2010, PLoS One, 5, e10412). Previously, we have demonstrated that intrabronchial inoculation of CPXV-Brighton Red (CPXV-BR) into cynomolgus monkeys resulted in a disease that shared many similarities to smallpox; however, severe respiratory tract disease was observed (Smith et al., 2011, J. Gen. Virol.). Here we describe the course of disease after small particle aerosol exposure of rhesus monkeys using computed tomography (CT) to monitor respiratory disease progression. Subjects developed a severe respiratory disease that was uniformly lethal at 5.7 log10 PFU of CPXV-BR. CT indicated changes in lung architecture that correlated with changes in peripheral blood monocytes and peripheral oxygen saturation. While the small particle aerosol inoculation route does not accurately mimic human smallpox, the data suggest that CT can be used as a tool to monitor real-time disease progression for evaluation of animal models for human diseases.

Poxvirus antigen staining of immune cells as a biomarker to predict disease outcome in monkeypox and cowpox virus infection in non-human primates.

Infection of non-human primates (NHPs) such as rhesus and cynomolgus macaques with monkeypox virus (MPXV) or cowpox virus (CPXV) serve as models to study poxvirus pathogenesis and to evaluate vaccines and anti-orthopox therapeutics. Intravenous inoculation of macaques with high dose of MPXV (>1-2×10(7) PFU) or CPXV (>10(2) PFU) results in 80% to 100% mortality and 66 to 100% mortality respectively. Here we report that NHPs with positive detection of poxvirus antigens in immune cells by flow cytometric staining, especially in monocytes and granulocytes succumbed to virus infection and that early positive pox staining is a strong predictor for lethality. Samples from four independent studies were analyzed. Eighteen NHPs from three different experiments were inoculated with two different MPXV strains at lethal doses. Ten NHPs displayed positive pox-staining and all 10 NHPs reached moribund endpoint. In contrast, none of the three NHPs that survived anticipated lethal virus dose showed apparent virus staining in the monocytes and granulocytes. In addition, three NHPs that were challenged with a lethal dose of MPXV and received cidofovir treatment were pox-antigen negative and all three NHPs survived. Furthermore, data from a CPXV study also demonstrated that 6/9 NHPs were pox-antigen staining positive and all 6 NHPs reached euthanasia endpoint, while the three survivors were pox-antigen staining negative. Thus, we conclude that monitoring pox-antigen staining in immune cells can be used as a biomarker to predict the prognosis of virus infection. Future studies should focus on the mechanisms and implications of the pox-infection of immune cells and the correlation between pox-antigen detection in immune cells and disease progression in human poxviral infection.

Cowpox virus employs a two-pronged strategy to outflank MHCI antigen presentation.

Smallpox decimated humanity for thousands of years before being eradicated by vaccination, a success facilitated by the fact that humans are the only host of variola virus. In contrast, other orthopoxviruses such as cowpox virus can infect a variety of mammalian species, although its dominant reservoir appears to be rodents. This difference in host specificity suggests that cowpox may have developed promiscuous immune evasion strategies to facilitate zoonosis. Recent experiments have established that cowpox can disrupt MHCI antigen presentation during viral infection of both human and murine cells, a process enabled by two unique proteins, CPXV012 and CPXV203. While CPXV012 inhibits antigenic peptide transport from the cytosol to the ER, CPXV203 blocks MHCI trafficking to the cell surface by exploiting the KDEL-receptor recycling pathway. Our recent investigations of CPXV203 reveal that it binds a diverse array of classical and non-classical MHCI proteins with dramatically increased affinities at the lower pH of the Golgi relative to the ER, thereby providing mechanistic insight into how it works synergistically with KDEL receptors to block MHCI surface expression. The strategy used by cowpox to both limit peptide supply and disrupt trafficking of fully assembled MHCI acts as a dual-edged sword that effectively disables adaptive immune surveillance of infected cells.

Structural mechanism of ER retrieval of MHC class I by cowpox.

One of the hallmarks of viral immune evasion is the capacity to disrupt major histocompatibility complex class I (MHCI) antigen presentation to evade T-cell detection. Cowpox virus encoded protein CPXV203 blocks MHCI surface expression by exploiting the KDEL-receptor recycling pathway, and here we show that CPXV203 directly binds a wide array of fully assembled MHCI proteins, both classical and non-classical. Further, the stability of CPXV203/MHCI complexes is highly pH dependent, with dramatically increased affinities at the lower pH of the Golgi relative to the endoplasmic reticulum (ER). Crystallographic studies reveal that CPXV203 adopts a beta-sandwich fold similar to poxvirus chemokine binding proteins, and binds the same highly conserved MHCI determinants located under the peptide-binding platform that tapasin, CD8, and natural killer (NK)-receptors engage. Mutagenesis of the CPXV203/MHCI interface identified the importance of two CPXV203 His residues that confer low pH stabilization of the complex and are critical to ER retrieval of MHCI. These studies clarify mechanistically how CPXV203 coordinates with other cowpox proteins to thwart antigen presentation.

Viral MHC class I inhibition evades CD8+ T-cell effector responses in vivo but not CD8+ T-cell priming.

Although viral MHC class I inhibition is considered a classic immune-evasion strategy, its in vivo role is largely unclear. Mutant cowpox virus lacking its MHC class I inhibitors is markedly attenuated during acute infection because of CD8(+) T-cell-dependent control, but it was not known how CD8(+) T-cell responses are affected. Interestingly, we found no major effect of MHC class I down-regulation on priming of functional cowpox virus-specific CD8(+) T cells. Instead, we demonstrate that, during acute infection in vivo, MHC class I down-regulation prevents primed virus-specific CD8(+) T cells from recognizing infected cells and exerting effector responses to control the infection.

Cowpox virus infection of cynomolgus macaques as a model of hemorrhagic smallpox.

Hemorrhagic smallpox was a rare but severe manifestation of variola virus infection that resulted in nearly 100% mortality. Here we describe intravenous (IV) inoculation of cowpox virus Brighton Red strain in cynomolgus macaques (Macaca fascicularis) which resulted in disease similar in presentation to hemorrhagic smallpox in humans. IV inoculation of macaques resulted in a uniformly lethal disease within 12 days post-inoculation in two independent experiments. Clinical observations and hematological and histopathological findings support hemorrhagic disease. Cowpox virus replicated to high levels in blood (8.0-9.0 log(10) gene copies/mL) and tissues including lymph nodes, thymus, spleen, bone marrow, and lungs. This unique model of hemorrhagic orthopoxvirus infection provides an accessible means to further study orthopoxvirus pathogenesis and to identify virus-specific and nonspecific therapies. Such studies will serve to complement the existing nonhuman primate models of more classical poxviral disease.

Cowpox virus induces interleukin-10 both in vitro and in vivo.

Cowpox virus infection induces interleukin-10 (IL-10) production from mouse bone marrow-derived dendritic cells (BMDCs) or cells of the mouse macrophage line (RAW264.7) at about 1800 pg/ml, whereas infections with vaccinia virus (strains WR or MVA) induced much less IL-10. Similarly, in vivo, IL-10 levels in bronchoalveolar lavage fluids of mice infected with cowpox virus were significantly higher than those after vaccinia virus infection. However, after intranasal cowpox virus infection, although dendritic and T-cell accumulations in the lungs of IL-10 deficient mice were greater than those in wild-type mice, weight-loss and viral burdens were not significantly different. IL-10 deficient mice were more susceptible than wild-type mice to re-infection with cowpox virus even though titers of neutralizing antibodies and virus-specific CD8 T cells were similar between IL-10 deficient and wild-type mice. Greater bronchopneumonia in IL-10 deficient mice than wild-type mice suggests that IL-10 contributes to the suppression of immunopathology in the lungs.