Development of HPV16 mouse and dog models for more accurate prediction of human vaccine efficacy.

BACKGROUND: Animal models are essential to understand the physiopathology of human diseases but also to evaluate new therapies. However, for several diseases there is no appropriate animal model, which complicates the development of effective therapies. HPV infections, responsible for carcinoma cancers, are among these. So far, the lack of relevant animal models has hampered the development of therapeutic vaccines. In this study, we used a candidate therapeutic vaccine named C216, similar to the ProCervix candidate therapeutic vaccine, to validate new mouse and dog HPV preclinical models. ProCervix has shown promising results with classical subcutaneous murine TC-1 cell tumor isografts but has failed in a phase II study. RESULTS: We first generated E7/HPV16 syngeneic transgenic mice in which the expression of the E7 antigen could be switched on through the use of Cre-lox recombination. Non-integrative LentiFlash® viral particles were used to locally deliver Cre mRNA, resulting in E7/HPV16 expression and GFP reporter fluorescence. The expression of E7/HPV16 was monitored by in vivo fluorescence using Cellvizio imaging and by local mRNA expression quantification. In the experimental conditions used, we observed no differences in E7 expression between C216 vaccinated and control groups. To mimic the MHC diversity of humans, E7/HPV16 transgenes were locally delivered by injection of lentiviral particles in the muscle of dogs. Vaccination with C216, tested with two different adjuvants, induced a strong immune response in dogs. However, we detected no relationship between the level of cellular response against E7/HPV16 and the elimination of E7-expressing cells, either by fluorescence or by RT-ddPCR analysis. CONCLUSIONS: In this study, we have developed two animal models, with a genetic design that is easily transposable to different antigens, to validate the efficacy of candidate vaccines. Our results indicate that, despite being immunogenic, the C216 candidate vaccine did not induce a sufficiently strong immune response to eliminate infected cells. Our results are in line with the failure of the ProCervix vaccine that was observed at the end of the phase II clinical trial, reinforcing the relevance of appropriate animal models.

Household Cases Suggest That Cats Belonging to Owners with COVID-19 Have a Limited Role in Virus Transmission.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for COVID-19 and spread rapidly following its emergence in Wuhan in 2019. Although cats are, among other domestic animals, susceptible to SARS-CoV-2 infection, little is known about their epidemiological role in the dynamics of a household infection. In this study, we monitored five cats for viral shedding daily. Each cat was confined with its COVID-19 positive owners in separate households. Low loads of viral nucleic acid were found in two cats, but only one developed anti-SARS-CoV-2 antibodies, which suggests that cats have a limited role in COVID-19 epidemiology.

Dynamic interactions between cephalexin and macrophages on different Staphylococcus aureus inoculum sizes: a tripartite in vitro model.

BACKGROUND: The bactericidal activity of an antimicrobial drug is generally assessed by in vitro bacterial time-kill experiments which do not include any components of the immune system, even though the innate immunity, the primary host defence, is probably able to kill a large proportion of pathogenic bacteria in immunocompetent patients. We developed an in vitro tripartite model to investigate the joint action of C57Bl/6 murine bone-marrow-derived macrophages and cephalexin on the killing of Staphylococcus aureus. RESULTS: By assessing the bactericidal effects on four bacterial inoculum sizes, we showed that macrophages can cooperate with cephalexin on inoculum sizes lower than 106 CFU/mL and conversely, protect S. aureus from cephalexin killing activity at the highest inoculum size. Cell analysis by flow cytometry revealed that macrophages were rapidly overwhelmed when exposed to large inoculums. Increasing the initial inoculum size from 105 to 107 CFU/mL increased macrophage death and decreased their ability to kill bacteria from six hours after exposure to bacteria. The addition of cephalexin at 16-fold MIC to 105 and 106 CFU/mL inoculums allowed the macrophages to survive and to maintain their bactericidal activity as if they were exposed to a small bacterial inoculum. However, with the highest inoculum size of 107 CFU/mL, the final bacterial counts in the supernatant were higher with macrophages plus cephalexin than with cephalexin alone. CONCLUSIONS: These results suggest that if the bacterial population at the infectious site is low, as potentially encountered in the early stage of infection or at the end of an antimicrobial treatment, the observed cooperation between macrophages and cephalexin could facilitate its control.

Single-cell analysis reveals new subset markers of murine peritoneal macrophages and highlights macrophage dynamics upon Staphylococcus aureus peritonitis.

Resident macrophages play a central role in maintaining tissue homeostasis and immune surveillance. Here, we used single cell-based qPCR coupled with flow cytometry analysis to further define the phenotypes of large and small resident peritoneal macrophages (LPMs and SPMs, respectively) in mice. We demonstrated that the expression of Cxcl13, IfngR1, Fizz-1 and Mrc-1 clearly distinguished between LPMs and SPMs subsets. Using these markers, the dynamics of peritoneal macrophages in a Staphylococcus aureus-induced peritonitis model were analyzed. We found that S. aureus infection triggers a massive macrophage disappearance reaction in both subsets. Thereafter, inflammatory monocytes rapidly infiltrated the cavity and differentiated to replenish the SPMs. Although phenotypically indistinguishable from resident SPMs by flow cytometry, newly recruited SPMs had a different pattern of gene expression dominated by M2 markers combined with M1 associated features (inos expression). Interestingly, S. aureus elicited SPMs showed a robust expression of Cxcl13, suggesting that these cells may endorse the role of depleted LPMs and contribute to restoring peritoneal homeostasis. These data provide information on both resident and recruited macrophages dynamics upon S. aureus infection and demonstrate that single-cell phenotyping is a promising and highly valuable approach to unraveling macrophage diversity and plasticity.

ANTIBODY RESPONSE TO EPSILON TOXIN OF CLOSTRIDIUM PERFRINGENS IN CAPTIVE RED DEER (CERVUS ELAPHUS) OVER A 13-MONTH PERIOD.

Deer are sensitive to clostridial diseases, and vaccination with clostridial toxoids is the method of choice to prevent these infections in ruminants. The purpose of this study was to evaluate the serologic responses in red deer (Cervus elaphus) over a 13-mo period after vaccination with a multivalent clostridial vaccine, containing an aluminium hydroxide adjuvant. Antibody production to the Clostridium perfringens type D epsilon toxin component of the vaccine was measured using an indirect enzyme-linked immunosorbent assay. Animals from group 1 (9 mo old; n = 6) were naïve and received an initial vaccination with a booster vaccine 4 wk apart and one annual booster. Animals from group 2 (21 mo old; n = 10) had been previously vaccinated 12 mo prior and received a first annual booster at the beginning of this study and a second annual booster 12 mo later. The multivalent clostridial vaccine induced a high antibody response that peaked after each injection and then slowly decreased with time. In group 1, a booster vaccine was required to obtain an initial high humoral response. The annual booster injection induced a strong, rapid, and consistent anamnestic response in both groups. The serologic responses persisted significantly over the baseline value for 9-12 mo in group 1, but more than 12 mo in group 2. It is unknown whether the measured humoral immune responses would have been protective as no challenge studies were performed. Further investigation is needed to determine the protective antibody titers to challenge and how long this immunity might persist after vaccination.

In vitro Degradation of Antimicrobials during Use of Broth Microdilution Method Can Increase the Measured Minimal Inhibitory and Minimal Bactericidal Concentrations.

The antibacterial activity of some antimicrobials may be under-estimated during in vitro microbiological susceptibility tests, due to their instability under such conditions. The in vitro degradation of seven widely used antimicrobials (amoxicillin, cephalexin monohydrate, cefotaxime sodium salt, ciprofloxacin, erythromycin hydrate, clarithromycin, and doxycycline hyclate) and its effect on MIC and MBC determinations was studied using the broth microdilution method, considered as the gold standard for MIC determinations. In vitro concentrations of antimicrobials, over a 24 h incubation period in the medium tested without bacteria, decreased from 33% for ciprofloxacin to 69% for clarithromycin. For cephalexin, cefotaxime, clarithromycin, and doxycycline which were the most degraded drugs, MIC and MBC values for one strain of E. coli and one strain of S. aureus were compared using the standard method or after ad-hoc drug complementation aiming at maintaining constant drug concentration. Abiotic degradation during the standard method was associated with a significant increase of the MIC (2 antibiotics) and MBC (3 antibiotics). However, the observed discrepancy (less than one twofold dilution), even for the most degraded drug for which the concentration at 24 h was reduced by two thirds, suggests that this would only be clinically significant in special cases such as slow-growing bacteria.

Pyroptosis of resident macrophages differentially orchestrates inflammatory responses to Staphylococcus aureus in resistant and susceptible mice.

The relationship between Staphylococcus aureus and innate immunity is highly complex and requires further investigation to be deciphered. i.p. challenge of C57BL/6 and DBA/2 mice, resistant and susceptible to the infection, respectively, resulted in different patterns of cytokine production and neutrophil recruitment. Staphylococcus aureus infection induced macrophage pyroptosis, an inflammasome-dependent cell death program, whose rates significantly differed between C57BL/6 and DBA/2 mice. Fast rate pyroptosis of C57BL/6 macrophages released high levels of IL-1ß but limited the synthesis of other cytokines such as TNF-α, IL-6, CXCL1, and CXCL2. Conversely, the extended survival of DBA/2 macrophages allowed substantial production of these NF-κB-related cytokines. Phenotyping of resting macrophages in different mouse strains revealed differential predisposition toward specific macrophage phenotypes that modulate S. aureus-mediated inflammasome activation. Treatment of DBA/2 susceptible mice with inflammasome inducers (i.e. nigericin and ATP) artificially increased pyroptosis and lowered the levels of NF-κB-related inflammatory cytokines, but restored IL-1ß to levels similar to those in C57BL/6 mice. Collectively, this study promotes the concept that, in association with host genetics, the basal phenotype of resident macrophages influences the early inflammatory response and possibly participates in S. aureus infection outcome via the inflammasome pathway and subsequent pyroptosis.

Transcriptomic analysis of milk somatic cells in mastitis resistant and susceptible sheep upon challenge with Staphylococcus epidermidis and Staphylococcus aureus.

BACKGROUND: The existence of a genetic basis for host responses to bacterial intramammary infections has been widely documented, but the underlying mechanisms and the genes are still largely unknown. Previously, two divergent lines of sheep selected for high/low milk somatic cell scores have been shown to be respectively susceptible and resistant to intramammary infections by Staphylococcus spp. Transcriptional profiling with an 15K ovine-specific microarray of the milk somatic cells of susceptible and resistant sheep infected successively by S. epidermidis and S. aureus was performed in order to enhance our understanding of the molecular and cellular events associated with mastitis resistance. RESULTS: The bacteriological titre was lower in the resistant than in the susceptible animals in the 48 hours following inoculation, although milk somatic cell concentration was similar. Gene expression was analysed in milk somatic cells, mainly represented by neutrophils, collected 12 hours post-challenge. A high number of differentially expressed genes between the two challenges indicated that more T cells are recruited upon inoculation by S. aureus than S. epidermidis. A total of 52 genes were significantly differentially expressed between the resistant and susceptible animals. Further Gene Ontology analysis indicated that differentially expressed genes were associated with immune and inflammatory responses, leukocyte adhesion, cell migration, and signal transduction. Close biological relationships could be established between most genes using gene network analysis. Furthermore, gene expression suggests that the cell turn-over, as a consequence of apoptosis/granulopoiesis, may be enhanced in the resistant line when compared to the susceptible line. CONCLUSIONS: Gene profiling in resistant and susceptible lines has provided good candidates for mapping the biological pathways and genes underlying genetically determined resistance and susceptibility towards Staphylococcus infections, and opens new fields for further investigation.

Simultaneous inactivation of espB and tir abrogates the strong, but non-protective, inflammatory response induced by EPEC.

Enteropathogenic Escherichia coli (EPEC) belong to the attaching and effacing (A/E) family of bacterial pathogens that represent a worldwide health concern. These non-invasive bacteria attach to intestinal enterocytes through a type III secretion system (T3SS), leading to intestinal inflammation and severe diarrhea. To dissect the signals leading to the induction of the inflammatory response and to understand its role in the pathogenesis of infection, we used the rabbit model, which represents a close model of human infections. Rabbits were orally inoculated with either the wild type O103:K-:H2 E22 EPEC strain or with the E22Δtir/espB strain, which bears mutations in two genes involved in the injectisome structure and function. To monitor the development of the inflammatory response, we developed a quantitative real-time RT-PCR (qPCR) assay specific for a panel of rabbit genes. Using combined immunohistochemistry and qPCR, we show here that the inflammatory response triggered by wild type EPEC occurs very early, preceding the bacterial colonization of the epithelium. However, this early response is unable to prevent bacterial attachment on enterocytes. Moreover, our results show that expression of a complete bacterial injectisome is required for the development of inflammation. Finally, infection by the virulent strain, but not by the doubly mutated strain, rapidly induces the development of a specific immune response in the mesenteric lymph nodes, which is not associated with protection. Our findings suggest that the induction of a strong inflammatory response by T3SS dependent components represents a selective advantage for T3SS+ bacteria, thereby facilitating their colonization.

Secretory IgA-mediated neutralization of Shigella flexneri prevents intestinal tissue destruction by down-regulating inflammatory circuits.

Shigella, a Gram-negative invasive enteropathogenic bacterium responsible for bacillary dysentery, causes the rupture, invasion, and inflammatory destruction of the human colonic mucosa. We explored the mechanisms of protection mediated by Shigella LPS-specific secretory IgA (SIgA), the major mucosal Ab induced upon natural infection. Bacteria, SIgA, or SIgA-S. flexneri immune complexes were administered into rabbit ligated intestinal loops containing a Peyer's patch. After 8 h, localizations of bacteria, SIgA, and SIgA-S. flexneri immune complexes were examined by immunohistochemistry and confocal microscopy imaging. We found that anti-Shigella LPS SIgA, mainly via immune exclusion, prevented Shigella-induced inflammation responsible for the destruction of the intestinal barrier. Besides this luminal trapping, a small proportion of SIgA-S. flexneri immune complexes were shown to enter the rabbit Peyer's patch and were internalized by dendritic cells of the subepithelial dome region. Local inflammatory status was analyzed by quantitative RT-PCR using newly designed primers for rabbit pro- and anti-inflammatory mediator genes. In Peyer's patches exposed to immune complexes, limited up-regulation of the expression of proinflammatory genes, including TNF-alpha, IL-6, Cox-2, and IFN-gamma, was observed, consistent with preserved morphology. In contrast, in Peyer's patches exposed to Shigella alone, high expression of the same mediators was measured, indicating that neutralizing SIgA dampens the proinflammatory properties of Shigella. These results show that in the form of immune complexes, SIgA guarantees both immune exclusion and neutralization of translocated bacteria, thus preserving the intestinal barrier integrity by preventing bacterial-induced inflammation. These findings add to the multiple facets of the noninflammatory properties of SIgA.

Protective role for protease-activated receptor-2 against influenza virus pathogenesis via an IFN-gamma-dependent pathway.

Protease-activated receptor-2 (PAR(2)), a receptor highly expressed in the respiratory tract, can influence inflammation at mucosal surfaces. Although the effects of PAR(2) in the innate immune response to bacterial infection have been documented, knowledge of its role in the context of viral infection is lacking. We thus investigated the role of PAR(2) in influenza pathogenesis in vitro and in vivo. In vitro, stimulation of PAR(2) on epithelial cells inhibited influenza virus type A (IAV) replication through the production of IFN-gamma. In vivo, stimulation of PAR(2) using specific agonists protected mice from IAV-induced acute lung injury and death. This effect correlated with an increased clearance of IAV in the lungs associated with increased IFN- gamma production and a decreased presence of neutrophils and RANTES release in bronchoalveolar fluids. More importantly, the protective effect of the PAR(2) agonist was totally abrogated in IFN- gamma-deficient mice. Finally, compared with wild-type mice, PAR(2)-deficient mice were more susceptible to IAV infection and displayed more severe lung inflammation. In these mice higher neutrophil counts and increased RANTES concentration but decreased IFN- gamma levels were observed in the bronchoalveolar lavages. Collectively, these results showed that PAR(2) plays a protective role during IAV infection through IFN-gamma production and decreased excessive recruitment of inflammatory cells to lung alveoli.

Safety and immunogenicity of myxoma virus as a new viral vector for small ruminants.

Myxoma virus (MYXV), a leporide-specific poxvirus, represents an attractive candidate for the generation of safe and non-replicative vaccine vectors for other species. With the aim of developing new recombinant vaccines for ruminants, we evaluated the safety and the immunogenicity of recombinant MYXV in sheep. In vitro studies indicated that ovine primary fibroblasts were not permissive for MYXV and that infection of ovine peripheral blood mononuclear cells occurred at a low rate. Although non-specific activation significantly improved the susceptibility of lymphocytes, MYXV infection remained abortive. Histological and immunohistochemical examination at the inoculation sites revealed the development of an inflammatory process and allowed the detection of sparse infected cells in the dermis. In addition, inoculated sheep developed an antibody response directed against MYXV and the product of the transgene. Overall, these results provide the first line of evidence on the potential of MYXV as a viral vector for ruminants.

Testing the efficacy of medium chain fatty acids against rabbit colibacillosis.

Enteropathogenic Escherichia coli (EPEC) represents a major cause of lethal diarrhea in young mammals. Although the pathogenicity mechanisms of EPEC are now well understood, the intrinsic and environmental factors that control the expression of EPEC virulence remain largely unknown. In the rabbit, suckling reduces pups' sensitivity to EPEC infection. Hence, we have hypothesized that uncharacterized factors present in doe milk may mediate this protection. Medium chain fatty acids (MCFA), known to possess antimicrobial properties, are highly abundant in doe milk. We demonstrate that caprylic acid exhibits a clear bacteriostatic effect in vitro against the rabbit EPEC strain E22 (O103:H2:K-), in a dose-dependent manner. In vivo, the dietary inclusion of triglycerides of MCFA did not however reduce the sensitivity of young rabbits challenged with this EPEC strain. The mortality and fecal excretion of EPEC were not reduced, and the bacterial adhesion to ileum was not inhibited. Amount of MCFA reaching the ileal level might have been too low and/or their association to other milk antimicrobials may have been required to observe a positive effect on disease evolution in a context of a highly virulent challenge.

Maternal milk contains antimicrobial factors that protect young rabbits from enteropathogenic Escherichia coli infection.

Enteropathogenic Escherichia coli (EPEC) colibacillosis represents a major cause of lethal diarrhea in young children in developing countries. EPEC strains also infect numerous mammal species and represent a major economical problem in rabbit industry. Protection against this pathogen is a challenging goal both in humans and in other mammal species. Despite a good knowledge of the pathogenicity mechanisms of EPEC, the intrinsic and environmental factors that control the expression of EPEC virulence in mammals remain unknown. For instance, the exacerbated sensitivity of young mammals to EPEC infection is still unexplained. Our goal was to investigate if age or other factors, like milk consumption, could be determinants that trigger the disease. We used rabbits as an animal model to study the role of milk in the sensitivity to an EPEC infection. Weaned and suckling rabbits were orally inoculated with EPEC strain E22 (O103:H2:K-) at 28 days of age, and the evolution of the disease was investigated in the two groups. In addition, in order to better characterize the interactions between milk and EPEC, we determined in vitro bacterial growth and the abilities of EPEC cells to adhere to epithelial cells in the presence of milk. Our results demonstrate a protective role of milk in vivo in association with in vitro antibacterial activity. These effects are independent of the presence of specific anti-EPEC antibodies.

The poxviral scrapin MV-LAP requires a myxoma viral infection context to efficiently downregulate MHC-I molecules.

Downregulation of MHC class I molecules is a strategy developed by some viruses to escape cellular immune responses. Myxoma virus (MV), a poxvirus causing rabbit myxomatosis, encodes MV-LAP that is known to increase MHC-I endocytosis and degradation through a C(4)HC(3) motif critical for an E3 ubiquitin ligase activity. Here, we performed a functional mapping of MV-LAP and showed that not only the C(4)HC(3) motif is necessary for a marked downregulation of MHC-I but also a conserved region in the C-terminal part of the protein. We also showed that the putative transmembrane domains are responsible for a specific subcellular localization of the protein: they retain MV-LAP in the ER in transfected cells and in the endolysosomal compartments in infected cells. We observed that a specific MV infection context is necessary for a fully efficient downregulation of MHC-I. Our data suggest that the functionality of viral LAP factors, inherited by herpes- and poxviruses from mammalian cells, is more complex than anticipated.

Genetically engineered enteropathogenic Escherichia coli strain elicits a specific immune response and protects against a virulent challenge.

Enteropathogenic Escherichia coli (EPEC), a major cause of severe disease with diarrhea in infants, is also involved in weaned rabbit colibacillosis. EPEC O103 is frequent in rabbit-fattening units of Western Europe. It causes high mortality and growth retardation, leading to substantial economic losses. We report here the construction by allelic exchange of an EPEC O103 strain mutated in espB and tir, two essential virulence genes. Upon live oral administration to weaned rabbits, the E22DeltaTir/EspB mutant strain efficiently colonized the intestinal tract without any adverse consequences. The rabbits were challenged with the highly pathogenic parental strain E22. The mutant provided complete protection to rabbits and total resistance to intestinal colonization by E22. In addition, E22DeltaTir/EspB strain induced a specific humoral response against the bacterial adhesin AF/R2. These Abs prevent bacterial attachment to epithelial cells in vitro. These results open the way for the development of an efficient vaccine strategy against rabbit EPEC infections.

New flow cytometric method to quantify the inhibition of enteropathogenic Escherichia coli adhesion by anti-adhesin antibodies.

BACKGROUND: Pathogenesis of enteropathogenic Escherichia coli (EPEC) infections can be divided in three stages. The first one is the intestinal colonization mediated by bacterial adhesins. The second and third stages are characterized by an intimate attachment of bacteria to the enterocytes. Little information is available on the specific immune response against EPEC. Here, we describe and validate a new approach to quantify the function of anti-EPEC adhesin antibodies (Abs). METHODS: We developed a new method to quantify the function of anti-adhesin Abs by flow cytometry. We used pEGFP-E22 (a rabbit EPEC E22 strain expressing the GFP protein) and HeLa cells. The adhesion of E22 bacteria to HeLa cells is mediated by AF/R2, the specific E22 adhesin. We performed short-time interaction (30 min) between pEGFP-E22 and HeLa cells. After extensive washes, 10,000 HeLa cells were acquired by flow cytometry and bacterial adhesion was quantified. Different sera were used to inhibit bacterial adhesion and recombinant MPB-Afr2G (Afr2G is the main AF/R2 subunit) was also tested in this system. RESULTS: We first verified that GFP expression by E22 did not modify bacterial adhesion. We then showed that this flow cytometry approach allowed easy quantification of bacterial adhesion and inhibition mediated by a specific anti-AF/R2 serum. Moreover, recombinant AF/R2 protein reversed the effect of the anti-AF/R2 serum. Finally, we validated our method using sera from E22 orally infected rabbits. We detected and quantified with this method functional specific anti-AF/R2 Abs in their sera. In addition, we correlated our results with an anti-AF/R2 enzyme-linked immunosorbent assay. CONCLUSIONS: We have developed a new method to detect and quantify specific anti-EPEC adhesin Abs by flow cytometry. This method is easy to use and highly reproducible. Its development could be extended to the search of specific anti-adhesin Abs in human EPEC infections.

A nonsecreted variant of interleukin-4 is associated with apoptosis: implication for the T helper-2 polarization in HIV infection.

We report the detection of an interleukin-4 (IL-4) variant whose expression is tightly associated with deprivation apoptosis. It is detected with the 8D4 anti-IL-4 monoclonal antibody (mAb) not only in T helper-2 (Th2) but also in Th1 clones, and primary T cells, and it is a nonsecreted molecule. It is not expressed during primary necrosis. Our data suggest that de novo IL-4 transcription of an alternative IL-4 mRNA (IL-4 delta(13)) is induced during deprivation apoptosis. In HIV-infected patients, increased expression of IL-4 in T cells is highly correlated to increased apoptosis, restricted to 8D4 reactivity (r(2) = 0.84 between % 8D4-8(+) and % 7- amino-actinomycin D-positive [7-AAD(+)] peripheral T cells, P <.0001), and associated with disease progression. The particular reactivity of apoptotic T cells to 8D4 mAb may explain some discordances among studies analyzing the Th1/Th2 balance in HIV infection and questions the function of this intracellular type 2 signal.

Myxoma virus leukemia-associated protein is responsible for major histocompatibility complex class I and Fas-CD95 down-regulation and defines scrapins, a new group of surface cellular receptor abductor proteins.

Down-modulation of major histocompatibility class I (MHC-I) molecules is a viral strategy for survival in the host. Myxoma virus, a member of the Poxviridae family responsible for rabbit myxomatosis, can down-modulate the expression of MHC-I molecules, but the viral factor(s) has not been described. We cloned and characterized a gene coding for an endoplasmic reticulum (ER)-resident protein containing an atypical zinc finger and two transmembrane domains, which we called myxoma virus leukemia-associated protein (MV-LAP). MV-LAP down-regulated surface MHC-I and Fas-CD95 molecules upon transfection; the mechanism probably involves an exacerbation of endocytosis and was lost when the ER retention signal was removed. In addition, the lytic activity of MHC-I-restricted antigen-specific cytolytic T lymphocytes (CTL) against myxoma virus-infected antigen-presenting target cells was significantly reduced, revealing a strong correlation between MHC-I down-regulation by MV-LAP and CTL killing in vitro. In vivo experiments with a knockout virus showed that MV-LAP is a virulence factor, potentially involved in the immunosuppression characteristic of myxomatosis. Data bank analysis revealed that MV-LAP has homologs in herpesviruses and other poxviruses. We propose the name "scrapins" to define a new group of ER-resident surface cellular receptor abductor proteins. The down-regulation of cell surface molecules by scrapins probably helps protect infected cells during viral infections.