T Cell Inactivation by Poxviral B22 Family Proteins Increases Viral Virulence
[['Smallpox was among the deadliest infectious diseases in history and its eradication is a landmark in medicine .', 'However , loss of orthopoxvirus ( OPXV ) -specific immunity facilitates the accidental introduction of zoonotic OPXV such as monkeypox virus ( MPXV ) and cowpox virus ( CPXV ) which cannot be eradicated due to animal reservoirs .', 'This risk became evident during the first MPXV outbreak outside Africa , which occurred in the US in 2003 [1] .', 'Although MPXV does not spread efficiently by human-to-human contact it shares several key features of pathogenesis with variola virus ( VARV ) the causative agent of smallpox .', 'MPXV is endemic in African rain forests with strains circulating in Central versus West Africa falling into two genetically distinct clades [2] .', 'The West African clade , including US2003 strains , is considered less virulent based on in vivo studies conducted in cynomolgus monkeys , prairie dogs , and ground squirrels [3] , [4] , [5] , [6] .', 'Nevertheless , life-threatening disease was identified during the U . S . outbreak [1] , [7] .', 'The DNA genomes of OPXV encode approximately 200 open reading frames ( ORFs ) with around 90 highly conserved genes encoded in the central regions of the genome whereas the terminally coded genes vary among different OPXV and are responsible for differences in host range , virulence , and immune evasion [8] .', 'Conserved genes among OPXV are highly related to each other resulting in cross-protection , i . e . prior infection with any one of the OPXV generally protects against serious disease by other OPX , so that vaccinia virus ( VACV ) is broadly protective against all OPXV .', 'Protection against OPXV is remarkably long lived .', 'During the 2003 MPXV outbreak , the number of lesions in previously vaccinated individuals was significantly lower with some individuals being completely protected from MPXV-associated disease [9] .', 'Antibody ( Ab ) titers to the vaccine remain remarkably stable over the life of vaccinated individuals [10] and vaccine-mediated protection of non-human primates ( NHP ) against lethal MPXV challenge is Ab-mediated [11] .', 'Similarly , vaccinated mice succumb to lethal challenge with mousepox ectromelia virus ( ECTV ) in the absence of Ab , despite the presence of poxvirus-specific T cells [12] .', 'In contrast , T cells promote survival of vaccinated mice challenged with lethal doses of VACV [13] , [14] .', 'The limited role of T cells in protecting against virulent OPXV is surprising given that OPXV induce a strong T cell response recognizing multiple conserved epitopes [15] .', 'Moreover , VACV is widely used as T cell-inducing vaccine vector [16] , [17] .', 'The reduced ability of T cells to control OPXV might , in fact , be directly related to virulence since T cells do limit virulence of CPXV provided that two gene products interfering with MHC-I antigen presentation were deleted [18] .', 'Thus , the inability of T cells in protecting against virulent OPXV might be due to T cell evasion mechanisms .', 'In the case of CPXV , T cell evasion is mediated by two gene products that each interferes with different steps of the MHC-I antigen presentation pathway .', 'CPXV203 binds to and retains MHC-I in the endoplasmic reticulum ( ER ) [19] .', 'CPXV12 inhibits TAP-dependent peptide translocation across the ER membrane [18] , [20] .', 'MPXV contains a CPXV203 orthologue , but does not seem to retain MHC-I [21] .', 'Instead , MPXV inhibits T cell activation by either MHC-dependent or by MHC-independent stimulation [21] .', 'Thus , MPXV encodes one or more gene products that render T cells non-responsive .', 'Here , we identify the gene product responsible for this T cell inactivation as MPXV197 , the largest gene in the MPXV genome .', 'This predicted transmembrane protein belongs to the B22 family of proteins found in several OPXV including CPXV , ECTV and VARV , but not in VACV .', 'We demonstrate that MPXV197 and related proteins of CPXV and VARV inactivate T cells by a novel mechanism .', 'We further show that deletion of MPXV197 severely attenuates MPXV and prevents lethal disease in rhesus macaques ( RM ) .', 'Despite a substantial reduction of viral titers , RM infected with MPXV197-deleted virus had stronger and more rapid T cell responses consistent with B22 proteins contributing to OPXV virulence by suppressing T cell responses .'], ['We previously demonstrated that MPXV ( Zaire strain ) inhibits CD4+ and CD8+ T-cell activation by both MHC-dependent and MHC-independent stimuli [21] .', 'In contrast , T cell evasion by CPXV seemed to rely predominantly on inhibition of MHC-I-dependent antigen presentation by CPXV12 and CPXV203 [18] , [19] , [20] , [22] .', 'Since MPXV Zaire encodes an orthologue of CPXV203 we wanted to determine whether it is required for T cell evasion .', 'The West-African strain MPXV US2003 lacks most of the CPXV203 orthologue [2] .', 'We compared poxvirus-specific T cell responses elicited by MPXV Zaire and US2003 by infecting human PBMC from recently VACV-vaccinated donors with MPXV at an MOI of 0 . 3 and analyzed T cell responses by intracellular cytokine staining ( ICCS ) for TNFα+ IFNγ+ cells .', 'VACV was used as control since it does not inhibit T cell stimulation [21] .', 'VACV vigorously stimulated virus-specific IFNγ+TNFα+ CD4+ and CD8+ T cells whereas ≤6% of either response occurred with MPXV Zaire or MPXV US2003 ( Fig . 1A ) .', 'Total IFNγ+ T cell responses or total TNFα+ T cell responses were reduced to the same level as that observed with IFNγ+TNFα+ T cells ( data not shown ) .', 'This lack of T cell stimulation was not due to lack of cross-reactivity or reduced rates of infection since VACV-specific T-cells recognize cells infected with UV-inactivated MPXV[21] and MPXV and VACV infected comparable numbers of cells in PBMC ( data not shown ) .', 'Thus , the MPXV homologue of CPXV203 is not required for inhibition of poxvirus-specific T cells .', 'To further examine whether the previously reported MHC-independent activation of T cells was also inhibited in the absence the CPXV203 orthologue we compared T cell activation by plate-bound αCD3 Ab in the presence of MPXV Zaire and US2003 .', 'As shown in Fig . 1A ( right panel ) , MPXV US2003 retained the capability to inhibit MHC- independent T cell stimulation .', 'T cell inhibitory genes are thus conserved in both clades of MPXV but absent or non-functional in VACV .', 'Since in PBMC , OPXV infect CD14+ monocytes but rarely T cells we concluded that T cell interference by MPXV is due to a trans-inhibition [21] .', 'However , to rule out with certainty that MPXV inhibits T cells in cis we separated MPXV-infection from antigen presentation by infecting human foreskin fibroblasts ( HFF ) with MPXV and co-incubating these cells with rhesus macaque ( RM ) -derived T cell lines specific for the MaMu-A*01-restricted SIV GAG181-189 epitope CM9 [23] .', 'As APC we used autologous B cells immortalized by simian lymphocryptovirus ( BLCLs ) [23] .', 'Thus , in this assay the infected cells ( HFF ) do not contribute to T cell stimulation which is provided by peptide-pulsed BLCLs .', 'To prevent dissemination of the virus from HFF to T cells we took advantage of the fact that compound ST-246 inhibits egress of viral particles from infected cells [24] .', 'Control experiments demonstrated that ST246 efficiently ( ∼90% ) prevented spread of VACV , CPXV , and MPXV to Jurkat T cells ( Fig . S1 ) .', 'When ST-246-pretreated HFF were infected with MPXV , T cell stimulation by CM9 peptide-pulsed BLCLs was still inhibited to <10% of the uninfected cell control ( Fig . 1B ) confirming that MPXV inhibits T cell activation in trans .', 'Since the T cell inhibitory factor is not secreted [21] this process most likely involves cell to cell contact .', 'To identify the T cell evasion gene ( s ) we generated four deletion mutants each lacking about 10 kb in the termini of the MPXV US2003 genome ( Fig . 1C; Fig . S2A ) .', 'Each of the mutants was examined for its ability to inhibit stimulation of T cells in PBMC from VACV-immune subjects ( Fig . 1D ) or peptide-stimulation of CM9-specific T cells from RM ( Fig . 1E ) .', 'Mutants lacking ORFs 11–25 , 26–35 ( data not shown ) , or 184–193 did not activate poxvirus-specific T cells ( Fig . 1D ) and still inhibited peptide-stimulation of CM9-specific T cells ( Fig . 1E ) .', 'In contrast , MPXV lacking ORFs 194–197 activated both CD4+ and CD8+ T cells in VACV-immune PBMC ( Fig . 1D ) and no longer inhibited peptide stimulation of CM9-specific T cells ( Fig . 1E ) .', 'These data suggested that the MPXV194–197 genomic region encodes the T cell inhibitor .', 'Since it seemed likely that T cell evasion would be mediated by a membrane bound extracellular protein , we deleted MPXV197 which is predicted to encode a large TM protein .', 'As shown in Fig . 1D and E , MPXVΔ197 stimulated poxvirus-specific CD4+ and CD8+ T cells similar to VACV and peptide stimulation of CM9-specific T cells was no longer inhibited .', 'Therefore , we conclude that MPXV197 is essential for T cell inhibition by MPXV .', 'To determine whether ectopically expressed MPXV197 inhibits T cell stimulation , we inserted a codon-optimized version into plasmid and adenovirus expression vectors .', 'MPXV197 is the largest ORF in the genome of MPXV encoding for 1880 amino-acids with a predicted molecular mass of 212 kDa , a predicted cleavable N-terminal signal peptide ( SP ) , multiple N-glycosylation sites , a C-terminal transmembrane ( TM ) domain , and potentially one or more internal TM domains ( Fig . 2A ) [2] .', 'Transient expression of MPXV197 in CHO cells and immunoblotting with αFLAG-Ab revealed two predominant bands with apparent molecular mass of ∼150 kDa , and ∼140 kDa and several minor , smaller bands as well as a large protein >250 kDa ( Fig . 2B ) .', 'To determine which of these proteins were located at the cell surface we performed surface biotinylation followed by streptavidin-precipitation and immunoblot with αFLAG antibody .', 'The ∼150 kDa species was the predominant species in this assay ( Fig . 2C ) .', 'Pulse-chase labeling revealed that the ∼150 kDa protein was a processing product derived from the large >250 kDa precursor protein .', 'A minor ∼140 kDa fragment carrying the C-terminal Flag-tag was synthesized simultaneously with the large precursor protein suggesting that this fragment is derived from an internal start site ( Fig . 2D ) .', 'Interestingly , Endoglycosidase H ( EndoH ) –treatment reduced the apparent molecular mass of the largest and the smaller fragment whereas the ∼150 kDa processing product was EndoH-resistant .', 'This result suggests that the full-length protein is processed into a ∼150 kDa fragment that is transported beyond the ER to the cell surface consistent with the surface biotinylation result .', 'The C-terminal location of the FLAG-tag identifies the ∼150 kDa fragment as a C-terminal fragment .', 'We further determined the sub-cellular localization of MPXV197 by immunofluorescence analysis ( IFA ) using confocal laser scanning microscopy ( CLSM ) .', 'Staining with αFLAG Ab of permeabilized or non-permeabilized CHO cells revealed that the C-terminus of MPXV197 locates to the extracellular face of the plasma membrane ( Fig . 2E ) .', 'In contrast , N-terminally Flag-tagged MPXV reacted with αFLAG Ab only when cells were permeabilized ( Fig . 2E ) consistent with the full-length protein and potential N-terminal fragments remaining intracellular .', 'The extracellular location of the C-terminus suggests that the C-terminal fragment most likely displays a multi-transmembrane topology with additional parts being exposed extracellularly ( Fig . 2A ) .', 'The fate of the remaining N-terminal part of MPXV197 is currently unknown and additional work will be required to delineate the topology of this protein in more detail .', 'To test whether MPXV197 inhibits T cell stimulation we co-incubated Ad-197 -transduced CHO cells with CM9-specific CD8+ T cells stimulated with peptide-pulsed BLCL .', 'Thus , T cells were stimulated by exposure to cognate peptides presented by BLCLs whereas MPXV197 is provided in trans by expression in CHO cells .', 'Since MPXV197 is under control of the tetracycline-regulated transactivator ( tTA ) CHO cells were co-transduced with Ad-tTA .', 'CHO cells transduced with Ad-tTA alone did not inhibit T cell activation with cognate antigen ( Ad-control , Fig . 3A ) .', 'In contrast , T cell responses were reduced to ∼0 . 01% of control upon MPXV197 expression ( Ad-197 , Fig . 3A ) .', 'Thus , MPXV197 inhibits T cell stimulation in trans even when provided by unrelated cells of a different species .', 'To measure the kinetics of the CM9-specific CD8+ T cell inactivation we co-incubated CM9-specific T cells with MPXV197-expressing cells for variable time periods prior to stimulation with peptide pulsed BLCLs .', 'T cell stimulation was reduced following as little as 1h of exposure to MPXV197 , with maximal inhibition at 6 h of co-incubation ( Fig . 3B ) .', 'Next we examined whether MPXV197 inhibits human T cell clones restricted by either classical ( HLA-B ) or non-classical ( HLA-E ) MHC-I using M . tuberculosis-specific CD8+ T cell clone D466 D6 recognizing peptide CFP2-12 presented by HLA-B [25] and D160 1-23 which is stimulated by pronase digested Mtb cell wall in the context of HLA-E [26] .', 'BEAS-2B epithelial cells were infected with either Ad-197 alone or together with Ad-tTA followed by incubation with Mtb-specific CD8+ T cell clones .', 'As shown in Fig . 3C , stimulation of both clones was inhibited by MPXV197 .', 'Since in this assay , MPXV197 is expressed in the same cells that present antigen we additionally examined antigen- and MHC-independent T cell stimulation of these T cell clones by phytohaemagglutinin ( PHA ) , a lectin that activates the TCR non-specifically by carbohydrate cross-linking .', 'PHA stimulation of both D466 D6 and D160 1–23 was inhibited by MPXV197 .', 'Taken together , these data demonstrate that MPXV197 recapitulates the T cell unresponsiveness mediated by MPXV for both human and non-human primate T cells regardless of the TCR stimulus .', 'Lack of cellular amine-reactive fluorescent staining ( LIVE/DEAD Fixable Dead Cell Stain ) indicates that T cell membranes remain intact in the presence of MPXV197 ( Fig . 3D , right panel ) .', 'To further determine whether T cells would be activated upon by-passing TCR stimulation we stimulated CM9-specific T cells with phorbol 12-myristate 13-acetate ( PMA ) which activates protein kinase C ( PKC ) and the Ca2+ ionophore ionomycin ( Iono ) .', 'Unlike peptide stimulation , MPXV197-expressing CHO cells did not inhibit T cell stimulation by PMA/Iono ( Fig . 3D , left panel ) .', 'Thus , T cells remain viable after exposure to MPXV197 suggesting that MPXV197 counteracts TCR-dependent signal transduction upstream of PKC .', 'Moreover , exposure of CM9-specific CD8+ T cells to MPXV197-expressing CHO cells did not impair their ability to bind a MaMu-A*01/CM9 tetramer suggesting that MPXV 197 does not interfere with MHC-I peptide loading ( Fig . 3E ) .', 'We conclude that MPXV197 interference occurs after TCR engagement with peptide/MHC complexes most likely inhibiting TCR proximal signal transduction .', 'MPXV197 belongs to the B22- protein family found in several OPXVs ( Fig . 4A ) including CPXV ( CPXV219 , 84% amino-acid identity ) , which causes zoonotic infections in humans , and VARV ( B22 , 86% amino-acid identity ) , the causative agent of smallpox .', 'To examine whether VARV B22 also inhibits T cell stimulation we inserted codon-optimized B22 encoding for 1897aa with a predicted molecular mass of ∼214 kDa into expression vectors .', 'Similar to MPXV197 , immunoblots and surface biotinylation of VARV B22 revealed surface expressed ∼150 kDa fragment with the B22 fragment being slightly larger than the corresponding MPXV197 fragment ( Figs . 4B , C ) .', 'Also similar to MPXV197 was the observation that the full-length precursor protein was barely detectable at steady state consistent with the 150 kDa protein being the final product .', 'The smaller protein bands were less abundant than those seen in MPXV197-expressing cells .', 'It is possible that these minor bands in both MPXV197 and VARV B22 are by-products of high level ectopic expression .', 'Similar to MPXV197 , we observed that the C-terminus of VARV B22 is exposed at the cell surface ( Fig . 4D ) .', 'T cell inhibition by VARV B22 was examined using both human Mtb-specific CD8+ T cell clones and rhesus CM9-specific CD8+ T cell lines .', 'As shown in Fig . 4E and F , VARV B22 inhibited T cell stimulation of both human and RM T cells as efficiently as MPXV197 .', 'These data strongly suggest that VARV inactivates T cells in a manner that is similar to MPXV .', 'To determine whether CPXV219 would similarly inhibit T cells we used a recombinant VACV expressing CPXV219 to infect BEAS-2B cells and examine stimulation of Mtb-specific T cells or to infect HFF and monitor stimulation of SIV-specific T cells by CM9 peptide loaded BCBLs .', 'Whereas VACV did not impact stimulation of human or RM T cells , VACV-219 inhibited T cell stimulation in both instances ( Fig . 5 A , B ) .', 'Thus it seems that the B22 proteins represent a family of T cell inhibitors .', 'The finding that CPXV219 inhibits T cells was unexpected since we previously reported that poxvirus-specific T cells were stimulated once MHC-I-dependent antigen presentation by CPXV was restored due to deletion of CPXV12 and CPXV203 [20] .', 'However , genome analysis of our deletion virus CPXVΔ12Δ203 revealed that , upon passaging , this mutant had acquired additional deletions downstream of CPXV203 due to a recombination event resulting in ORF204-221 being replaced by a duplication of ORF10-11 ( data not shown ) .', 'Therefore , this deletion virus ( now designated CPXVΔ12Δ203-221 ) lacks not only CPXV12 and CPXV203 , but also CPXV219 .', 'However , an independently generated CPXVΔ12Δ203 mutant was also reported to stimulate poxvirus-specific T cells [18] , although this analysis was limited to murine T cells .', 'To determine the impact of CPXV219 on human and mouse T cells we analyzed CPXV mutants lacking CPXV219 alone or together with CPXV12 and CPXV203 and mutants lacking CPXV12 and CPXV203 .', 'Stimulation of poxvirus-specific human T cells was analyzed by infecting PBMC from VACV-immune subjects with CPXV and monitoring T cell activation by ICCS whereas stimulation of murine T cells was monitored by adding splenocytes from VACV-immunized mice to CPXV-infected A20 cells ( Fig . 5C , D ) .', 'CPXV did not stimulate poxvirus-specific human CD8+ and CD4+ T cells whereas mutant A694 lacking the genomic region CPXV204-221 stimulated human CD4+ T cells but not CD8+ T cells ( Fig . 5C ) .', 'Since A694 contains CPXV12 and CPXV203 these data suggest that human CD8+ T cells are not stimulated due to MHC-I evasion whereas human CD4+ T cells were stimulated due to the absence of CPXV219 .', 'Indeed , CPXVΔ12Δ203-221 lacking CPXV219 as well as CPXV12 and CPXV203 restored stimulation of both human CD4+ and CD8+ T cells .', 'In contrast , CPXVΔ12Δ203 did not restore stimulation of poxvirus-specific human CD8+T cells ( Fig . 5C ) , despite the restoration of MHC-I presentation [18] .', 'These results are consistent with CPXV219 inhibiting both human CD4+ and CD8+ T cells in a manner similar to MPXV197 .', 'However , when stimulation of murine poxvirus-specific T cells was examined with the same series of mutants , CD8+ T cells were stimulated in the absence of CPXV12 and CPXV203 even when CPXV219 was present ( Fig . 5D ) as reported [18] .', 'Interestingly however , CPXVΔ12Δ203 showed reduced activation of CD4+ T cells compared to VACV or CPXVΔ12Δ203-221 suggesting that CPXV219 does not efficiently inactivate murine CD8+ T cells but might impact murine CD4+ T cells .', 'Together with the finding that MPXV stimulated murine CD8+ T cells ( data not shown ) these results indicate that B22 proteins inhibit human and monkey T cells , but are less active against murine T cells .', 'Using a rabbit anti-serum raised against purified GST-tagged CPXV219 we examined its expression in CPXV-infected human 143 cells and CHO cells as well as in HEK 293 cells infected with VACV-219 ( Figs . 5E , F ) .', 'CPXV219 was expressed with early kinetics and detectable as early as 3h p . i . ( data not shown ) .', 'Metabolic pulse/chase labeling and immunoprecipitation at 3 h p . i . further demonstrated that a high molecular mass product ( >220 kDa ) was processed into a ∼150 kDa fragment ( Fig . 5E ) .', 'Consistent with the ∼150 kDa fragment being the final product , a similarly sized protein was the predominant fragment in immunoblots of CPXV-infected CHO cells whereas this was absent from CPXVΔ219-infected cell lysates ( Fig . 5F ) .', 'Similarly , a ∼150 kDa fragment was the predominant protein found in VAC-219 infected cells in the absence of the T7 polymerase .', 'However , upon co-infection with T7-polymerase expressing VACV , the >250 kDa precursor was highly expressed whereas the ∼150 kDa fragment was only slightly increased consistent with the majority of the protein remaining in the ER-resident precursor state upon overexpression .', 'Taken together with the data shown above for MPXV197 and VARV B22 , these data suggest that in both virally infected and ectopically expressing cells the full-length precursor protein is processed into a ∼150 kDa fragment .', 'Since the anti-CPXV219 antiserum was raised against the whole protein , it is not known which part of the protein is recognized .', 'However , the ∼150 kDa fragment of both MPXV197 and VARV B22 was detected by a C-terminal FLAG-tag suggesting that the CPXV219 ∼150 kDa fragment is likewise C-terminal .', 'Thus , we conclude that a ∼150 kDa C-terminal fragment is the ultimate product of MPXV197 , VARV B22 and CPXV219 and that this fragment is transported to the cell surface where it acts as T cell inactivator .', 'Since B22 proteins are more active against primate than rodent T cells we used a recently described intrabronchial ( i . b . ) inoculation model in RM [27] to determine the role of MPXV197 in viral dissemination , pathogenesis and induction of T cell responses .', 'To rule out that MPXVΔ197 contained additional mutations compared to parental strain MPXV-US2003 we sequenced the genomes for both viruses by next generation ( NextGen ) sequencing .', 'Within a margin of error ( <3% ) both WT and MPXVΔ197 matched the predicted sequence exactly ( Fig . S3 , Table S2 ) .', 'Since this analysis cannot distinguish between sequencing errors , misalignments ( particularly in the repeat region ) and actual mutations , it is likely that the actual percentage of correct genome sequences is substantially higher .', 'Therefore , we conclude that the vast majority of genomes present in our WT control and MPXV197-deleted virus contain the expected genome sequence .', 'We infected 8 RM with MPXV-US2003 or MPXVΔ197 using i . b . inoculation of 2×105 PFU ( Fig . 6A ) , a dose at which MPXV-Zaire was non-lethal [27] .', 'The clinicopathologic course of infection was followed by physical examination , biotelemetry to record body temperature and activity , O2 tissue saturation , and development of cutaneous lesions .', 'Blood and bronchoalveolar lavage ( BAL ) fluid samples were collected at defined days post infection ( dpi ) to determine the kinetics of virus replication and of the adaptive immune response .', 'As shown in Fig . 6 and table S3 , RM infected with MPXVΔ197 experienced a significantly shorter duration of fever ( 5 days compared to 20 days ) ( Fig . 6B ) , fewer skin lesions ( Fig . 6E ) , and dramatically reduced morbidity and mortality .', 'In fact , two of the MPXV-US2003-infected RM had to be euthanized due to deteriorating health whereas all four of the MPXVΔ197-infected RM spontaneously controlled the infection prior to termination of the experiment at days 41 and 42 .', 'Viral titers measured in the lungs were initially similar , reflecting the similar size of the inoculum , but lung titers of MPXVΔ197 fell significantly more rapidly compared to WT ( Fig . 6C ) .', 'An even more striking contrast was observed for viral titers in the blood where all RM infected with MPXV-US2003 showed significantly higher levels of viremia compared to MPXVΔ197 which was barely detectable ( Fig . 6D ) .', 'Interestingly , while uncontrolled viremia in both lungs and blood correlated with rapid deterioration of health in one animal ( WT-4 ) , the other animal that needed to be euthanized prematurely ( WT-3 ) had a lower viremia in the blood but a higher number of lesions at days 14 and 21 compared to the remaining WT-infected RM ( Fig . 6E ) .', 'In contrast , low titers in the blood correlated with a generally mild disease and less than 30 lesions in MPXVΔ197-infected RM ( Fig . 6E , Table S3 ) .', 'Decreased viral titers of MPXVΔ197 were also reflected in a decrease of antibody titers which tended to be lower than that of MPXV-US2003 although this was not statistically significant ( Fig . 6F ) .', 'In stark contrast to the reduced virologic and disease parameters , poxvirus-specific T cell responses were detected earlier and were significantly higher at some of the earliest time points in RM infected with MPXVΔ197 compared to MPXV-US2003 ( Fig . 7A ) .', '( Note that T cell responses were measured using VACV to avoid the T cell inhibitory effect of MPXV197 ) .', 'At day 14 , all four MPXVΔ197-infected RM had a significantly higher frequency of poxvirus-specific CD8+ T cells in their blood compared to the 3 remaining WT-infected RM ( Fig . 7A ) .', 'Similarly , in 3 of 4 MPXVΔ197-infected RM the CD4+ T cell response was above background at days 7 and 14 whereas 0/4 or 2/3 WT-infected RM had detectable CD4+ and CD8+ T cells at these days .', 'At day 21 , the frequency of CD4+ T cells in all MPXVΔ197-infected RM was significantly higher than in WT-infected RM .', 'The inverse correlation between viral titers and T cell responses in the blood is consistent with MPXV197 contributing to viral dissemination during the early phase of infection by delaying the onset of the cellular immune response .', 'To examine whether the T cell inactivation mediated by MPXV197 would result in a systemic suppression of T cell responses during viral infection in vivo , we stimulated T cells in PBMC with αCD3 Ab .', 'The data is limited to three WT and two MPXVΔ197-infected RM since two animals were missing samples and T cells from Δ197-3 was unresponsive to αCD3 stimulation potentially due to CD3 polymorphism .', 'Although the overall frequency of T cells in the blood did not change during infection ( Fig . 7B ) , there was a dramatic reduction in αCD3 responses of both CD4+ and CD8+ T cells from WT-infected RM at 7–21 dpi ( Fig . 7C ) .', 'This was particularly evident at day 14 which correlated with peak viremia in the blood of WT-1 and WT-2-infected animals ( Fig . 6D ) .', 'In contrast , this decrease was less pronounced for αCD3-stimulation of T cells in both MPXVΔ197-infected RM .', 'Although not statistically significant due to the low sample size , these observations are consistent with MPXV197 contributing to a systemic suppression of T cell responses during peak viremia .'], ['We report here a novel mechanism by which viral proteins inhibit T cell responses and describe the impact of this immunomodulation on viral virulence and immunity .', 'The activation of T cells via TCR engagement with peptide/MHC complexes is the principal mechanism by which CD8+ T cells recognize and eliminate virus-infected cells and by which CD4+ T cells recognize APC .', 'To limit T cell control , viruses can thus either interfere with antigen presentation or they can interfere with the ability of T cells to respond to antigen .', 'Many instances of the former mechanism have been described , particularly for large DNA viruses that are limited in their ability to escape from T cell control by mutating immunodominant epitopes [28] .', 'However , MHC-inhibitors are unable to prevent activation of T cells by non-infected APC processing exogenous antigens via MHC-II presentation or by MHC-I cross-presentation .', 'In contrast , B22 proteins would allow poxviruses to interfere with TCR-dependent T cell activation regardless of the stimulatory pathway .', 'While indirect inhibition of T cell stimulation , e . g . by interference with cytokine networks [29] , [30] , [31] , [32] has been described for poxviruses , very few instances of viruses directly inhibiting T cells have been reported to date .', 'Among poxviruses , a secreted CD30 homologue of ECTV was shown to inhibit CD4+ T cells activation in mixed lymphocyte reactions [33] .', 'However , the CD30 homologue encoded by the CPXV strain used in our studies did not seem to affect T cell stimulation since both human and mouse T cells were activated upon elimination of the MHC-I inhibitory genes CPXV12 and CPXV203 and the T cell inactivator CPXV219 .', 'It was also reported that VACV inhibits TCR-dependent responses of γδ T cells [34] , but this inhibitory mechanism clearly does not apply to αβ T cells since we used VACV as our control in all T cell experiments .', 'To our knowledge , the most closely related mechanism described so far for a viral protein interfering with T cell stimulation was reported for UL11 of human cytomegalovirus ( HCMV ) which binds to CD45 on the surface of T cells and inhibits TCR-mediated signaling [35] .', 'However , UL11 is unable to prevent the stimulation of HCMV-specific T cells by fibroblasts infected with HCMV lacking viral MHC-I inhibitors [36] .', 'In contrast , deletion of CPXV MHC-I inhibitors did not restore human T cell stimulation when CPXV219 was present ( Fig . 5C ) .', 'Moreover , MPXV seems to rely solely on MPXV197 for T cell inhibition .', 'Thus , the strong inhibitory effect of B22 proteins stands out since it seems to require only a brief interaction with T cells , even non-cognate T cells , to shut down T cell stimulation .', 'Since effector T cells are exquisitely sensitive to activating signals , one peptide/MHC complex reportedly can activate cytokine secretion in T cells [37] , the T cell inhibition by B22 family proteins is truly remarkable .', 'This silencing of T cells is likely transient since T cells respond to stimuli that by-pass TCR signaling such as PMA/Ionomycin in the presence of MPXV197 ( Fig . 3D ) .', 'The B22 inhibitory impact could thus be best described as transient inactivation , or transient “functional paralysis” , a term previously suggested for HCMV UL11 [35] .', 'The molecular mechanism by which B22 proteins inactivate T cells is currently unknown .', 'A multitude of proteins expressed on T cells can exert negative signals , in many cases overriding the ability of the TCR to signal .', 'Engagement of these inhibitory receptors results in peripheral T cell tolerance that can be either permanent , as in T cell exhaustion , or temporary .', 'Therefore , these proteins are often referred to as immune checkpoint proteins or co-inhibitory proteins [38] .', 'Conceivably , B22 proteins could exert their effect by engaging one or several of these co-inhibitory proteins .', 'Since B22 proteins seem to be unable to block murine CD8+ T cells it seems that the target protein on T cells is either primate-specific or that it is differentially expressed on T cells in mice .', 'The inability of these proteins to inhibit murine T cells might be the reason why this effect has not been observed previously when studying CPXV in mice .', 'Since ECTV is a mouse pathogen it will be interesting to examine whether the ECTV B22 homologue is capable of inhibiting mouse T cells .', 'If not , the question arises whether B22 proteins perform a different function in rodents , or whether the lack of T cell inhibition is only observed in the genus mus , but not in other rodent species .', 'Since both CPXV and MPXV infect rodents in the wild , it would be interesting to explore the species-specificity of this protein family in more detail .', 'Additionally , B22 proteins might perform different functions in different species .', 'For instance it is conceivable that in addition to the membrane bound ∼150 kDa C-terminal fragment , a secreted amino-terminal fragment is generated that has functions other than T cell inactivation .', 'In contrast to MPXV and CPXV which display a rather wide host range , VARV is highly restricted to humans whi
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Infections with monkeypox , cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population , but little is known about their mechanisms of virulence and immune evasion .', 'We now demonstrate that B22 proteins , encoded by the largest genes of these viruses , render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation .', 'In contrast , stimuli that bypass TCR-signaling are not inhibited .', 'In a non-human primate model of monkeypox , virus lacking the B22R homologue ( MPXVΔ197 ) caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia , morbidity and mortality .', 'Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003 , we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination .']
['We discovered that the largest gene in the genome of monkeypox viruses and several related viruses , including the virus causing smallpox , but not vaccine strains , encode a protein ( B22 ) that renders the cellular immune system non-responsive .', 'A particularly novel aspect of this work is that B22 proteins directly disable cells of the immune system as compared to previously known molecular strategies that help viruses to hide from the immune system .', 'We further show that monkeypox viruses containing this protein are much more virulent in non-human primates than viruses that lack B22 .', 'Our observations suggest that B22 proteins contribute to monkeypox virulence and might have contributed to the severe disease manifestations of variola major virus .', 'However , these data also suggest that B22 proteins could potentially be used to curb undesired immune responses such as autoimmunity or graft versus host disease .']
['biology and life sciences', 'immunology']