These either involve the omission of do, or non-target-like movement of a lexical verb. Furthermore, Emma also allows verb movement across the subject with both lexical verbs and auxiliaries in topicalised structures, suggesting that she has overgeneralised residual V2 across verb types and clause types. Emily, on the other hand, is very target-like in structures involving residual V2 in English, but also allows auxiliaries and dummy-do to move across the subject in topicalised structures, overgeneralising residual V2 to apply to non-subject-initial declaratives. Finally, Sunniva is very precocious and very target-like in all the relevant structures, which may be an indication of acceleration due to CLI from Norwegian V2. We discuss these results with reference to language balance, finding that the measures available to us suggest that the differences between the children cannot straightforwardly be explained by language dominance. Instead, we suggest that these results can be accounted for by ambiguity in the English system, leaving the data open to several possible interpretations when acquired in contact with the consistent V2 system in Norwegian. This has several consequences: (i) the three girls’ parsers interpret the input differently, (ii) differences between the three children are qualitative rather than quantitative and (iii) there has to be some mechanism that ensures that the children can ‘recover’ from these non-target-like grammars. In this paper, we will focus on the first two issues.This paper investigates the acquisition of residual verb second (V2) in three corpora consisting of data from Norwegian-English bilinguals  in order to determine to what extent these structures are affected by cross-linguistic influence (CLI) from Norwegian V2. The three girls exhibit three different patterns with regard to the relevant constructions. They are very target-like in their use of auxiliaries in the relevant structures. However, when it comes to  Moreover, in interrogative clauses, English displays Subject Auxiliary Inversion (SAI) (2). However, in both negative and interrogative clauses, do-support is required in the absence of a finite auxiliary . In non-subject-initial declaratives, so-called topicalised constructions, English does not display SAI (5).The relevant contexts we investigate in this paper are illustrated in (1)–(5). English has V-to-T movement with auxiliaries and (1)have not seen her.I (2)Have you seen her?(3)did not buy the book.I (4)Did you buy the book?(5)have bought a new car.(a) Today I       (b) Yesterday I watched the new Star Wars movie.do-support and non-subject-initial clauses in English that all diverge from what is typically found in monolingual English-speaking children. We will therefore explore whether all these three outcomes of the bilingual situation are due to CLI from Norwegian V2.The three bilingual girls in this study display three different patterns with respect to the acquisition of residual V2, do-support and verb placement in English and Norwegian. We highlight where the two languages display superficially overlapping surface structures, and where the two systems are underlyingly  different, thus pointing out where CLI due to structural overlap might be expected. In the ‘Previous Research on the Acquisition of Auxiliaries and Do-Support in English’ section, we present previous research on do-support and verb placement in monolingual English-speaking children, as well as previous research on the acquisition of verb placement in children acquiring English alongside a (Germanic) V2 language. Finally, in the ‘Research Questions and Predictions for the Current Study’ section, we present the research questions of the current paper. In the ‘Materials and Methods’ section, we introduce the three bilingual girls studied in this investigation, as well as our methodology. In the ‘Results’ section, we present the results of our investigation. In the ‘Discussion’ section contains a discussion of the results, and we explore to what extent the differences between the three girls can be attributed to language dominance. In the ‘Conclusion’ section concludes the paper.The paper is structured as follows. In the ‘Background’ section, we first provide the relevant background on target-like In this section, we outline the crucial background on verb placement in Norwegian and English, highlighting areas of superficial structural overlap that might be susceptible to CLI.Norwegian is an SVO language, and as a result, the verb will generally precede the object (6).(6)så bilen.Jeg I saw car.the            ‘I saw the car’.Furthermore, like its Germanic relatives (except English), it is also a V2 language. This means that the finite verbal element moves to the second position in all main clauses har ikke sett bilen.(a) Jeg I have not seen car.the                      ‘I haven’t seen the car’.så ikke bilen.      (b) Jeg I saw not car.the                      ‘I didn’t see the car’.yes/no-questions in  and wh-questions , as well as in topicalised structures (9), V2 leads to inversion of the finite verb and the subject.1Moreover, interrogatives, illustrated by (8)Har du sett den?(a) Have you seen it                      ‘Have you seen it?’Så du den?     (b) Saw you it                      ‘Did you see it?’har du kjøpt?     (c) Hva What have you bought                      ‘What have you bought?’kjøpte du?     (d) Hva What bought you                      ‘What did you buy?’(9)har jeg kjøpt en bil.(a) Idag Today have I bought a car                      ‘Today I have bought a car’.kjøpte jeg en bil.      (b) Idag Today bought I a car                      ‘Today I bought a car’.Like Norwegian, English is an SVO language, as illustrated in (10).(10)saw the car.I auxiliaries will precede negation and adverbs, as in (11a). In the absence of a finite auxiliary, the phenomenon of do-support emerges in negative declaratives.In contrast, however, English is not a V2 language but exhibits residual V2. This is a reflection of the fact that modern day English has remnants of a grammatical system that used to be more like the one observed in other Germanic languages today, where the finite verbal element typically was the second constituent in the clause. V2 in modern English is residual in two ways. While the other Germanic languages exhibit V2 in all clause types [cf. (6)–(9) above], V2 only applies in certain clause types in English. Moreover, while any finite verb has to move to the second position in Norwegian, V2 only applies to a subset of verbs in English, viz. auxiliaries. Consequently, only finite (11)have not seen the car.(a) I did not see the car.        (b) I yes/no-questions  and wh-questions , residual V2 leads to inversion of the finite auxiliary and the subject . Again, in clauses without a finite auxiliary, do-support is required :Moreover, in (12)Have you seen it?(a) Did you see it?        (b) have you bought?        (c) What did you buy?        (d) What do-support in the second position in these contexts:Finally, in topicalised structures, neither finite main verbs nor finite auxiliaries undergo movement across the subject in English. Furthermore, there is no requirement for (13)I have bought a car.(a) Today I bought a car.        (b) Today here and there.2Note, however, that remnants of V2 can be found in certain topicalised structures, for example, clauses introduced by short adverbials such as Crucially, when such structures contain full DP subjects they trigger V2-like structures , while with pronominal DP subjects they occur without V2 .(14)comes the bride.(a) Here she comes.        (b) Here (15)’s Noddy.(a) Therehe is.        (b) There Even though these structures are infrequent in English, they are relevant in this context because they provide evidence to the learner of a V2 grammar in English.yes/no-questions and in wh-questions the two languages display finite auxiliaries in parallel positions (17).As mentioned in the ‘Introduction’ section, it has been argued that areas where the two languages in a bilingual situation display superficial structural overlap are particularly vulnerable to CLI. When looking at word order and verb placement in particular in English and Norwegian, there are several similarities. Both languages are SVO (16). Moreover, in negative declaratives, in (16)så bilen.Norwegian: Jeg saw the car        English: I (17)har ikke sett bilen.(a) Norwegian: Jeg have not seen the car             English: I Har du sett den?        (b) Norwegian: have you seen it?             English: har du kjøpt?        (c) Norwegian: Hva have you bought?             English: What here/there. This yields the same word order as in Norwegian:In addition, English has verb movement of finite auxiliaries across the full DP subject in clauses introduced by adverbials such as (18)kommer bruden.Norwegian: Her comes the bride.        English: Here do-support in these contexts (19).However, when the finite verbal element is a lexical verb, rather than an auxiliary, the overlap breaks down. In Norwegian, lexical verbs also move to the second position in negative and interrogative clauses, while English employs (19)så ikke bilen.(a) Norwegian: Jeg ∗I saw not the car             English: did not see the car             I Så du den?       (b) Norwegian: ∗Saw you it?             English: Did you see it?             kjøpte du?       (c) Norwegian: Hva ∗What bought you?             English: did you buy?             What In addition, the two languages show distinct patterns in non-subject-initial declaratives, where again, Norwegian has a consistent V2 pattern, while English has no verb movement to the second position (20):(20)har jeg kjøpt en bil.(a) Norwegian: Idag ∗Today have I bought a car.English:              I have bought a car.             English: Today kjøpte jeg en bil.       (b) Norwegian: Idag ∗Today bought I a car.English:              I bought a car.             English: Today We will argue that the superficial structural overlap shown above may lead to CLI.do-support in English focusing on negative declaratives and interrogatives. Then we review some studies on verb placement in bilingual children acquiring English as one of their languages.In this section, we first briefly address previous research on the acquisition of auxiliaries and be, which on average is first attested at 2;2 in her data. A couple of months later, at 2;7, the first use of auxiliary be is found. The age of the first use of do-support is on average 2;8, while auxiliary have is the last, and only attested in Stromswold’s data as late as at 3;5. See also be, do and auxiliary be. They do not discuss auxiliary have.It is well known that children go through an early stage in which they systematically omit functional elements marking tense and agreement. However, finite verbs are rarely completely absent from child grammars at this stage  (17.2%). Notably, with respect to age, there does not seem to be any significant differences; both groups contain children within the whole age range from 2;5 to 3;4. This suggests that there is a lot of variation concerning at what age productive do-support in negative declaratives is acquired.For negative structures, the developmental path has been argued to involve an initial stage of pre-sentential negation, such as wh-questions at an early stage  up to the age of at least 2;9. In somewhat contrast to this, wh-questions and yes/no-questions (as well as declaratives) more than 80% of the time.Turning to interrogatives, several studies have shown that auxiliaries tend to be omitted in ly stage . In a stwh-questions, yes/no-questions and wh-questions show variable results. Some studies do not find differences between the two types of interrogative clauses  or in declaratives as a superfluous do in non-emphatic contexts (24b)  from : 92, 136(21)want not some water. I (22)make the kittens? What (23)throw daddy in the water. The flower (24)did get this from? (a) Where I did watch it.         (b) I wh-questions displayed the patterns in (21) and (22) in the early stage . As pointed out in the previous section, monolingual English-speaking children hardly ever produce this kind of verb movement. Knipschild argues that the non-target-like utterances in (21)–(23) above are due to transfer from German. Similar findings in the English of bilingual German/English children have been reported by In fact, more than 90% of negated clauses and do-support, this is only employed in negative declaratives between the ages of 2;0 and 2;11 and in questions from the age of 3;0.In a case study of an Icelandic/English bilingual girl Katla, As highlighted in the ‘Verb Placement in Norwegian and English’ section, there is considerable superficial structural overlap between Norwegian and English, suggesting that CLI can be expected. Moreover, studies of bilingual children acquiring English together with other Germanic V2 languages reveal that such influence does occur. Given this, our research questions are as outlined in (1)–(3), and we make the predictions in (4) and (5).(1)do-support in English affected by simultaneous acquisition of Norwegian?To what extent is the acquisition of residual V2 and (a)all verb types, including lexical verbs?Is residual V2 expanded to apply to (b)all clause types, including topicalised structures?Is residual V2 expanded to apply to (c)all verb types and all clause types, resulting in a full V2 system?Is residual V2 expanded to both (d)do-support delayed or accelerated?Is the acquisition of residual V2 and especially (2)Are all the three children affected by CLI from Norwegian in the same way?(3)If they are not, what can explain the differences?(4)do-support in bilingual children, the following logical possibilities exist:If CLI from V2 in Norwegian affects the acquisition of residual V2 and (a)to all verb types, including lexical verbs:If residual V2 is expanded to apply (i)do-support should be delayed, as it makes the phenomenon superfluous in the grammar, andthe acquisition of (ii)lexical verbs should occur in the position normally reserved for auxiliaries in questions and negative declaratives.(b)to all clause types, SAI and do-support should also occur in topicalised structures.If residual V2 is expanded to apply (c)to all verb types and all clause types, the children should allow a full V2 grammar.If residual V2 is expanded to apply (d)do-support, these phenomena should be attested at an earlier stage in bilingual children.If CLI from Norwegian V2 accelerates the acquisition of residual V2 and (5)Given that there is a great deal of ambiguity in the English system, it should be possible for different parsers to be affected by simultaneous input from Norwegian V2 in different ways, resulting in different grammars.(6)If such differences occur, they can be explained as an effect of language dominance.4The current study is a corpus study based on data from three girls, all bilingual from birth: Emma, Sunniva and Emily. These corpora were collected by the authors in connection with previous projects.As mentioned, the three girls grew up in very similar language situations; they all have one native English-speaking parent and one native Norwegian-speaking parent and grew up in Tromsø, Norway. Thus, English is a heritage language and Norwegian is the majority language in the lives of these children. The Norwegian-speaking parents opted to speak English with their children as well as with their English-speaking spouses. Thus, in all three cases, English is the home language. All girls attended nursery from around the age of one, Emily slightly later as she was born in the summer and started after the summer holiday, at approximately 14 months. Thus, this is the age at which consistent exposure to Norwegian started, even though both families were in close contact with family, friends and society at large, making some exposure to Norwegian likely most days even before the age of 1.Two of the children, Emma and Sunniva, were also the first child in the family, while Emily has two older siblings, one of them being Sunniva. Emma’s English-speaking parent is her American mother, while Sunniva and Emily’s father is British. Sunniva and Emily also speak English with one another and with their brother. There is a 10-year age difference between the two sisters.W). Emma was recorded biweekly in both English and Norwegian in the course of a three-month period between the ages of 2;7.10 and 2;10.9. There are six English files in Emma’s corpus, consisting of 1831 child utterances. In these files, her MLUW range is 3.074–3.998. Sunniva was recorded in English and Norwegian for approximately a year, from age 1;6.25–2;8.0, at irregular intervals. There are nine files and 2512 utterances in her English data. Her MLUW ranges from 1.992–3.667 in these files. The equivalent information about Emma’s and Sunniva’s Norwegian files can be found in Table W range is 2.833–4.961, with the first two recordings clustering between 2.8 and 3 and the last two ranging from 4.7 to almost 5.The data from Emma, Sunniva and Emily form the basis of the current study. Relevant information about the three corpora is summarised in Table do-support or another auxiliary were identified: (i) negative declaratives, (ii) yes/no-questions and (iii) wh-questions. We included (iv) non-subject-initial declaratives in the searches, some of which also involve auxiliaries or do-support .Emma’s files were originally transcribed by the Norwegian investigator and were later checked by a native speaker of English. Sunniva’s and Emily’s files were transcribed by a native speaker of English and subsequently checked by a native speaker of Norwegian. For the current study, the files from the three children were searched manually for the relevant structures. In the searches, all the contexts that obligatorily involve wh-question. However, there are some exceptions to this. First, repetitions were included when the child kept repeating the same sentence but produced it in different forms [e.g. (26) below]. Similarly, when the child repeated an adult utterance incorrectly, the relevant example would be included in the count, but not when the child only repeated a part of the utterance (27).Direct repetitions, both of other interlocutors and self-repetitions, were generally excluded. For example, the two examples in (25) were only counted as one (25)Sunniva: Where’s the pillow? Where’s the pillow?                Mother: Where’s the pillow?        Mummy can’t see the pillow from here.(26)Emily: Where are the cats gone?         Where the cats gone?Where the cats gone?                Mummy?        Mother: Hmm?        Emily: You have to say: Where are the cats gone?        Mother: Where have the cats gone?        Emily: Here.(27)Sunniva: What do you got?         Mother: It’s a little mouse, I think.Sunniva: Little mouse, I think.        Finally, identical repetitions produced by the child because she was explicitly asked to do so by the adult interlocutor were also included in the count. Other structures that were excluded from the counts were utterances that were questioned in the transcription or for which alternative transcriptions were proposed, both indicating that the transcriber was unsure about the relevant utterance. Similarly, when a central part of the utterance is incomprehensible, the relevant example was not included in the count .(28)Mother: Mummy gonna put the (.) knickers on the little dolly.         Mother: On this little dolly.        Mother: I think they go on like this.Sunniva: Where [?] xxx the knickers gone?                Mother: Huh?In other situations, examples where the incomprehensible part did not have any consequences for the relevant phenomena were included. Structures involving utterances where the transcriber was unsure about the transcription or where central parts of it were incomprehensible were double checked with the sound files and included or excluded depending on whether the authors agreed, disagreed or were still unsure about the relevant utterances.5.As Table (29)Can you sit on my back? (30)’s not broken mummy. Winnie the Pooh(31)’s that here on your watch? What(32)not doing it. I TARGET: I’m not doing it.        (33)I’m drawing, mummy? What TARGET: What am I drawing?        do-support, on the other hand, the situation is very different, especially for Emma. While 86.5% of her questions and negative structures include the auxiliary, do-support is only employed in 16.7% of structures requiring this. Furthermore, even though the majority of non-target-like utterances simply lack do (50%), similarly to what has been observed for monolingual English children, close to a third of Emma’s questions and negative structures displays movement of a lexical verb (33.3%). In comparison, Emily and Sunniva supply do at a very similar rate to other auxiliaries, at 92.6 and 91.2%, respectively. These results are summarised in Table For contexts requiring cal verb 3.3%. In (34)did you make [/] make some waffles? Where (35)do you want to see? Cinderella, (36)don’t watch mmm dance competition. No, I (37)hurt not this knee now. I TARGET: I did not hurt this knee now        (38)not know. I TARGET: I don’t know        (39)Drive daddy me to barnehage? TARGET: Will/did daddy drive me to nursery?        do-support in the three children.Considering these examples in more detail, we see that Emma in general is less target-like than Emily and Sunniva with all the structures involving residual V2. Tables yes/no-questions (78.5%) than in wh-questions (63.6%) and negative declaratives (47.7%). However, as shown in Table yes/no-questions (47/65) involve an auxiliary other than do, and these are all target-like. Of the 18 yes/no-questions requiring do-support, only 22.2% are target-like. All the non-target-like yes/no-questions involve movement of the lexical verb. As Table do (73.3%). Only a small proportion exhibit lexical verb movement (14.6%). The two other children are more consistent (and target-like) across the various structures.As these tables show, Emma is more target-like in tions 78.% than inuestions 3.6% and here/there . There are only two relevant examples attested in her corpus, and both of these are target-like. Both Emma and Emily produce topicalisations with verb movement. However, while Emma allows verb movement of both auxiliaries and lexical verbs, Emily only exhibits verb movement of auxiliaries. Furthermore, these two girls make use of verb movement at very different rates. As Table 6Turning to non-subject-initial structures, we see that all three children are very different from one another. As shown in Table (40)then did Belle go up again. And (41)Then you need to take some shampoo. (42)Now throw I it. (43)Now I step on the scary woman. wh-questions, yes/no-questions and negation. With respect to yes/no-questions, Emma is also target-like in her use of auxiliaries, while she is somewhat less consistent in wh-questions and negative declaratives. However, when it comes to structures that require do-support, Emma is much less target-like that Emily and Sunniva. In this section, we discuss the results in more detail, and address the research questions posed in ‘Research Questions and Predictions for the Current Study’. Recall that the main research questions concerned (1) to what extent the acquisition of residual V2 and do-support in Norwegian-English bilinguals is influenced by Norwegian V2, (2) whether all the three children are affected in the same way and (3) if they are affected differently, what can explain the differences.So far, we have seen that both Emily and Sunniva are quite target-like in their use of auxiliaries in do-support is obsolete. A second possible outcome of CLI could be the expansion of residual V2 to all clause types, resulting in consistent SAI in topicalised declaratives. CLI might also affect both verb types and clause types, resulting in (the possibility of) a full V2 grammar in the children’s English. A final possibility is that the simultaneous acquisition of Norwegian V2 might accelerate the acquisition of residual V2 and especially do-support. The reasoning behind this prediction is that Norwegian V2 word order may enhance the need for a verbal element in a pre-negation position in negative declaratives and in a pre-subject position in interrogative clauses.In what follows, we consider each of the three children in turn with regard to possible CLI from Norwegian. In doing so, we also consider whether the children are affected by this possible influence in the same way, or whether different parsers could possibly interpret the bilingual input in different ways. Recall that we predicted that CLI might take several forms. It might cause residual V2 to be expanded to include all verb types, which would result in lexical verbs preceding the subject in questions and non-subject-initial clauses and preceding negation in negative declaratives. A by-product of such influence could be that do-support is required. As shown in Table do-support (73.2%). In addition, 14.6% of negative declaratives requiring do-support instead displays non-target-like movement of the lexical verb, as illustrated by the example in (44). Moreover, Table yes/no-questions involve non-target-like verb movement of the lexical verb, see (45) below:As we saw in Table (44)hurt not this knee now. I (45)Drive daddy me to barnehage (=daycare)? gonna (not included in Tables Gonna (going to) is not a lexical verb but patterns with lexical verbs with regard to placement in negative declaratives and questions, as illustrated in (46). However, in Emma’s negative declaratives, gonna almost exclusively occurs in front of the negation  (47):These examples indicate CLI from Norwegian V2 across verb types in Emma’s data. Another indication of this is demonstrated by Emma’s placement of (46)not gonna make it.He’s (47)gonna not sleep more. Now that Furthermore, there are also indications of CLI from Norwegian V2 across clause types in Emma’s data. As revealed by Table (48)throw I it. Now (49)have I ringed Angus. Now Thus, Emma meets predictions (4a)–(4c). Due to influence from Norwegian V2, V2 appears to be transferred to apply across verb types and across clause types in Emma’s English, making it almost equivalent to her Norwegian grammar in this respect.do-support is also very target-like. Recall that she supplies auxiliaries in questions and negative structures in the target position at 95.1% (135/142) and do in the same structures at 92.6% (65/68). There is only one example of V2 with a lexical verb. Thus, it seems clear that Emily has not expanded residual V2 to apply to lexical verbs [thus not meeting prediction (4a)]. However, as illustrated in Table do-support in such contexts at 31.6% (24/76), as illustrated in (50) and (51), making as much as 65.8% (50/76) of topicalised clauses non-target-like.As shown in Table (50)was madame Gazelle’s telephone ringing. And then (51)did Candy Cat come. And then Thus, we argue that there is CLI from Norwegian V2 into English also in Emily’s data, causing residual V2 to be expanded across clause types, confirming prediction (4b).do-support, which is included in 91.2% of required contexts (31/34) , nor does she produce any non-target-like topicalisations [but notably she only produces two (non-imitated) non-subject-initial structures] (52).Finally, Sunniva is also target-like and consistent with respect to her use of auxiliaries and copula, including such elements 93.2% of the time (164/176), as shown in Table (52)he’s swimming. Maybe It would thus appear that Sunniva is not affected by CLI in residual V2 structures, contrary to predictions (4a)–(4c).do-support to be accelerated. There are some challenges with respect to this issue. For one thing, the three children investigated in the current study are in relatively different age spans. Recall from Table This leaves prediction (4d), suggesting that CLI from V2 may cause the acquisition of residual V2 and do-support has been observed to occur in negation before questions in both monolingual and bilingual children. In our data, there is a total of 10 examples of do-support in all of Emma’s files, five in negative structures, four in yes/no-questions and one in wh-questions. In Emily’s first two files, aged 2;3.25 and 2;4.19, she produces 29 instances of do-support, four of which are in questions, which at least does not seem to be late compared to monolinguals. In Sunniva’s files, however, there are 12 instances of do-support before the age of two; all but one occur in negative structures. Both types are illustrated in (53) and (54).Furthermore, as discussed in the ‘Previous Research on the Acquisition of Auxiliaries and Do-Support in English’ section, (53)don’t watch mmm dance competition. No, I (54)did you make [/] make some waffles? Where do-support at a very young age, it is difficult to say for sure whether this is (i) early compared to monolinguals and (ii) early compared to Sunniva’s general development.Even though Sunniva clearly makes use of do-support in a negative clause is attested at age 2;0, and then there are several (11) at age 2;2. In questions, the first example is attested at age 2;2, and then two at age 2;3 and eleven at age 2;5. A quick search of three (randomly selected) children in the Manchester corpus ]. Then the remaining eleven examples attested before the age of 2;0 are produced at 1;11.22. Thus, with regard to the question of first attestations, Sunniva does not seem that different from the three monolinguals from the Manchester corpus or Susan reported in do-support than the three Manchester children at an early stage in development. In the files at ages 1;11.01, 1;11.29 and 2;0.26, which comprise a total of 2677 child utterances, Joel makes use of do-support in four instances (0.15%). Anne produces 10 examples among the 4339 utterances she produces at ages 1;10.07, 1;11.06 and 1;11.20 (0.23%). Finally, Aran makes use of do-support eight times among his 4139 utterances at ages 1;11.12, 2;0.09 and 2;1.07 (0.19%). In comparison, Sunniva’s 12 examples are uttered in the course of the 1300 utterances she produces at ages 1;6.25, 1;9.13, 1;10.01 and 1;11.22 (0.92%). Thus, even though one from this cannot definitively conclude that she has acquired do-support earlier than these monolingual peers, it seems fair to say that she uses do-support more than these monolingual children at this early stage of development.With regard to the first question, r corpus , Anne, Ado-support is simply a reflection of her generally being a precocious speaker, there are some indications that this might be the case. In the three early files in Joel’s data , his MLUW ranges from 1.299 to 1.846. Anne’s MLUW  is between 1.558 and 2.233, while Aran’s  is 1.299–2.341. In comparison, Sunniva’s four early files  have MLUW between 1.992 and 3.168, which is considerably higher than the three monolinguals, not just in absolute terms, but also in relation to her age. Sunniva’s MLUW is also high in comparison with the other two bilinguals, as illustrated in Table Regarding the question of whether Sunniva’s early use of do-support can be shown to follow different paths in the three bilinguals in the current study. Emma exhibits CLI across verb types and across clause types, and thus shows a behaviour compatible with predictions (4a)–(4c). Emily transfers across clause types and makes use of residual V2 and do-support in non-subject-initial declaratives, confirming prediction (4b). The data from these two children put together show the validity of predictions (4b) and (4c). Emily’s behaviour demonstrates that residual V2 may be expanded to apply to topicalised structures (4b), thus filling a gap in English in terms of how non-subject-initial structures pattern [cp. (8) and (9) to (12) and (13) in the ‘Verb Placement in Norwegian and English’ section]. Emma’s grammar, however, expands residual V2 into general V2 both in terms of verb types and clause types and thus is in accordance with prediction (4c). However, based on these results, we cannot be sure whether bilingual acquisition in this context could lead to CLI across verb types only (4a), resulting in a grammar where all verb types, including lexical verbs, may precede the negation in negative declaratives and subjects in questions, but not in topicalisations. Sunniva, on the other hand, does not exhibit any kind of transfer and is very precocious in her use of do-support. However, as she also appears to have a higher MLUW in relation to her age than both the three monolinguals we have compared her to and the other bilinguals in the current study, this is most likely not due to accelerated development as a result of CLI. Consequently, we cannot draw any conclusions regarding prediction (4d). However, these results together confirm prediction (5), as the simultaneous exposure to the V2 language Norwegian and residual V2 in English seems to result in different developmental paths for the acquisition of residual V2 and particularly do-support. Thus, it appears that different parsers may interpret the input differently in bilingual situations such as these. The question is what exactly might cause this to happen, specifically whether language dominance can explain the observed differences.The development of residual V2 and especially The final research question addresses to what extent the differences between the three children can be explained with reference to language dominance, the underlying assumption being that CLI is more likely to occur from the dominant to the weaker language  level of proficiency in the two languages and/or the (relative) exposure to and use of the two languages  topicalised structures. Thus, Sunniva potentially may have gone through a period after data collection finished when she exhibited the same behaviour as Emily.However, one possible explanation for the differences between the sisters might simply be that the data that we have available do not capture the period when Sunniva exhibits the same behaviour as Emily. Recall that Emily’s non-target-like topicalised structures occur in the later files , while there are no non-subject-initial declaratives in Emily’s early files .W underdetermines the score in Swedish compared to Italian because the Swedish definite article is suffixal, while the Italian one is a free morpheme. Consequently, only the latter would be included in an MLUW count.8 Note that the same difference applies between the two languages investigated here, Norwegian and English (bil-en versus the car). From this perspective, a higher MLUW is expected in English, all other things being equal. Apart from this specific fact, the two languages are relatively similar with regard to morphological complexity and the realisation of various functional elements as free or bound.When proficiency has been used as an indicator of dominance, many different types of measures may be used to determine the balance between the two languages. MLU  is frequently been used in corpus studies , while Sunniva’s average for English is 2.833 and for Norwegian 2.648. On this measure, both Emma and Sunniva would be classified as ‘strongly balanced’ according to W may to underdetermine the score for Norwegian compared to English because of the different status of definite article in the two languages. Nevertheless, with respect to MLUW, both Emma and Sunniva are very balanced.An overview of the three children’s MLUAnother possible indicator of dominance is language mixing, as the direction of mixing often is claimed to occur from the stronger to the weaker language cf. . We onlyCorrelating language mixing as a measure of language dominance and balance, we would expect both Emily and Emma to have a higher proportion of mixing in their English than Sunniva, possibly also with Emma mixing somewhat more than Emily (on the assumption that dominance can be gradient). However, as we can see from Table (55)ordentlig on egentlig. (Emily)It wasn’t TARGET: It wasn’t properly on really.        (56)Nå skal dolly sove. (Sunniva)TARGET:Now the dolly is going to sleep.        (57)en tå. (Emma)I falled on TARGET: I fell on a toe.        do-support. Emma, the child who is the most influenced by Norwegian, mixes less than Sunniva, who appears to be the most target-like with respect to English verb placement. However, another factor also pertains to who the interlocutor is in the different files. In Emma’s English files, she is mostly with her American mother, while in her Norwegian files, she is playing with an investigator whom she believes does not speak English. Sunniva’s English files mostly include her Norwegian (but English-speaking) mother, while the Norwegian files are recorded with a Norwegian investigator. Note, however, that the mother is almost always present. Emily falls in between Sunniva and Emma in terms of acquisition of SAI and do-support but mixes the most of all of them. Emily’s (English) files mainly include her Norwegian (but English-speaking) mother. One relevant question is why Emily mixes more than the other two. A factor contributing to this might be that Emily’s older brother code-switches quite extensively, and in the context of a family where all the members are very fluent in both languages, code-switching is thus a natural communicative strategy also for Emily.These results thus reveal two things: (i) both Sunniva and Emma, the two children we have both English and Norwegian files from, mix more in their English than in the majority language and (ii) the proportion of mixing in the children’s English does not seem to be correlated with the extent to which they behave target-like with residual V2 and Further arguments against an explanation in terms of language dominance are provided in other studies involving Sunniva and Emma. Ws in English and Norwegian, and they mix more Norwegian into their English than the other way around. For Emily, we do not have access to Norwegian data, and thus cannot compare her English and her Norwegian competence. However, overall, she is not particularly delayed in her acquisition of English, which one might expect if Norwegian was strongly dominant. Furthermore, the rate of mixing does not seem to reflect the extent to which the three girls are target-like in their behaviour. Finally, the fact that English is their home language combined with frequent exposure to the community language supports the impression of three bilinguals who are very balanced. This also means that prediction (6) is not confirmed. The differences between the children cannot be explained with reference to language dominance. Rather, it seems that the three children (at least Sunniva and Emma) behave differently despite being relatively similar with regard to language balance. In the next section, we explore to what extent the different behaviours can be accounted for with reference to the two linguistic systems.To sum up, it appears that all the measures of dominance available to us indicate that Emma and Sunniva are fairly balanced bilinguals. Both of them have very similar MLUdo-support, the three children diverge. While Emily and Sunniva are very target-like also in these contexts, Emma produces a high proportion of non-target-like utterances. Recall from ‘Results’ section that unlike the other two, Emma employs verb movement of lexical verbs across the negation in negative structures and across the subject in yes/no-questions, suggesting that she has overgeneralised residual V2 to apply across verb types. Furthermore, she also allows both SAI and verb movement of lexical verbs in non-subject-initial declaratives, suggesting that she has overgeneralised V2 to apply across clause types as well, thus confirming predictions (4b) and (4c). For Emily, we have seen that even though her behaviour is very target-like in structures involving residual V2, she overgeneralises auxiliary movement to topicalised structures, and as a result, the majority of her non-subject-initial declaratives involve non-target-like SAI or do-support. We have further seen that language dominance, at least as it can be measured with these data, cannot explain the differences between the children. Nevertheless, we observe that the parsers of the three children somehow interpret the data differently.So far, we have seen that the three bilingual children investigated in the current study behave very target-like when it comes to auxiliary placement in negative structures and questions. However, in contexts requiring Do-Support in English’ section that even though English does not make use of SAI or verb movement in non-subject-initial declaratives, adverbials such as here and there in initial position may cooccur with V2 [cf. (14) and (15)]. One possible explanation for Emily and Emma’s non-target-like behaviour with topicalisations is that they have been exposed to a large number of these structures in the input, with a high proportion involving DP subjects, causing them to overgeneralise V2 into topicalisations in English. If this is the case, we would expect Emma and Emily to have had more exposure to and make more use of such structures than Sunniva. However, as illustrated by Table here/there is not completely in line with what the adult speakers in the same corpus do. Notably, Emma makes use of these topics more than her mother (19 versus 12), while Emily is the one with the highest number of these structures . The distribution of ±V2 is also quite similar to that of the adults, but slightly more skewed towards V2. Emma is the one with the clearest preference for V2 in these structures (89.5%), but these are all target-like. It thus seems that the frequency of these structures in the input cannot account for the variation among the children (even though these corpora clearly are very limited).Recall from ‘Residual Verb Second and here and there. As demonstrated in Table here/there are topicalised. The first two columns in the table show the children’s use of V2 (+V2) with DP subjects and V3 (-V2) with pronouns when the topic is here or there. As the table reveals, the children follow this pattern completely. Then the next six columns show the same distribution  with other topics  and with lexical verbs, auxiliaries and copula be and do, respectively. Note that in these columns any structure that is +V2 is ungrammatical, but the closer the percentage in each of these columns is to 100%, the more similar the child’s behaviour with topics in general is to here/there. As we can see, Emily consistently has V2 with DP subjects, except with lexical verbs, which she does not allow in the V2 position. With pronominal subjects, her behaviour is more variable, but clearly a substantial amount of her pronominal subjects also occurs with V2 , thus going against the pattern. Emma exhibits a high preference for both V2 with DP subjects  and for V3 with pronominal ones . What is surprising is that it is the two children who appear to have been exposed to these structures the least who have adopted the word order pattern with V2. One possible explanation for this might be that the children need exposure to a certain number of examples to realise that structures such as non-subject-initial declaratives with here/there actually represent an exception. In the absence of sufficient exposure, the parser makes an overgeneralisation based on the available data, which for Norwegian/English bilinguals also will include data with massive indications of V2. Moreover, V-to-T movement of auxiliaries in English causes negative declaratives to superficially look similar to Norwegian constructions involving V2. The same is true for positive declaratives with adverbials such as often and always when they include an auxiliary. In the absence of auxiliaries, however, the similarity breaks down. This division between auxiliaries and lexical verbs makes the English system more ambiguous than the Norwegian one, leaving it open to several possible interpretations.9 According to do).Interestingly, however, an investigation into the children’s behaviour with DP versus pronominal subjects in non-subject-initial declaratives suggests that the two children who overgeneralise residual V2 are indeed influenced by the word order variation found with do-support is caused by CLI from Norwegian. Furthermore, we have discussed various possible explanations for the differences between the three children’s acquisition of verb placement in English. It is not obvious that the differences between the children can be explained with reference to language dominance, nor can they be explained in terms of frequency of exposure to non-subject-initial structures exhibiting optional V2. We have suggested that the observed CLI can be accounted for by the ambiguity in the English system, which leaves the data open to several possible interpretations when English is acquired in contact with the consistent V2 system in Norwegian. It thus seems that the children’s parsers may interpret the input differently. Importantly, this means that the differences between the children are qualitative rather than quantitative. Furthermore, for Emily, we also know that she was able to ‘recover’ from this grammar, and we assume the same is true for Emma (who is an adult now), suggesting that this kind of recovery has to be possible and needs to be accounted for in developing grammars. We leave to future research the question of how such recovery from a non-target-like grammar is possible.This paper investigates the acquisition of residual V2 in three Norwegian-English bilinguals. We find that the three girls exhibit three different patterns with regard to the relevant constructions, despite the fact that they grew up in comparable language situations. We argue that the non-target-like behaviour with respect to verb placement and This research is based on child language corpora collected in a period from 1999 to 2012. Parents were thoroughly informed about the data collection and the purpose of this, and signed consent forms on behalf of themselves and the children we collected data from. At the point in time when these data were collected, there were no requirements of approval from an ethics committee in Norway, and thus, such approval has not been obtained specifically for the collection of these data. However, approval has been obtained from NSD – Norwegian Centre for Research Data for the overall research project MiMS  which the current investigation is a part of.All authors listed have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CP and handling Editor declared their shared affiliation.


Cochliobolus carbonum race 1 (CCR1). The first identified case of APR in maize is encoded by a hypomorphic allele, Hm1A, at the hm1 locus. In contrast, wild-type alleles of hm1 provide complete protection at all developmental stages and in every part of the maize plant. Hm1 encodes an NADPH-dependent reductase, which inactivates HC-toxin, a key virulence effector of CCR1. Cloning and characterization of Hm1A ruled out differential transcription or translation for its APR phenotype and identified an amino acid substitution that reduced HC-toxin reductase (HCTR) activity. The possibility of a causal relationship between the weak nature of Hm1A and its APR phenotype was confirmed by the generation of two new APR alleles of Hm1 by mutagenesis. The HCTRs encoded by these new APR alleles had undergone relatively conservative missense changes that partially reduced their enzymatic activity similar to HM1A. No difference in accumulation of HCTR was observed between adult and juvenile plants, suggesting that the susceptibility of seedlings derives from a greater need for HCTR activity, not reduced accumulation of the gene product. Conditions and treatments that altered the photosynthetic output of the host had a dramatic effect on resistance imparted by the APR alleles, demonstrating a link between the energetic or metabolic status of the host and disease resistance affected by HC-toxin catabolism by the APR alleles of HCTR.Adult plant resistance (APR) is an enigmatic phenomenon in which resistance genes are ineffective in protecting seedlings from disease but confer robust resistance at maturity. Maize has multiple cases in which genes confer APR to northern leaf spot, a lethal disease caused by  hm1 gene at different stages of development. Using a combination of molecular genetics, biochemistry and physiology, we present multiple lines of evidence that demonstrate that APR is a feature or symptom of weak forms of resistance. While the mature parts of the plant are metabolically robust enough to manifest resistance, seedling tissues are not, leaving them vulnerable to disease. Growth conditions that compromise the photosynthetic output of the plant further deteriorate the ability of the seedlings to protect themselves from pathogens.Adult plant resistance (APR) is a phenomenon in which disease resistance genes are able to confer resistance at the adult stages of the plant but somehow fail to do so at the seedling stages. Despite the widespread occurrence of APR in various plant diseases, the mechanism underlying this trait remains obscure. It is not due to the differential transcription of these genes, and here we show that it is also not due to the differential translation or activity of the APR alleles of the maize  Plant responses to pathogens are dynamic, and they involve a number of inducible mechanisms that are tightly regulated both in space and time . They arLr34 gene-mediated resistance, in which the onset against the leaf rust pathogen, Puccinia triticina, is largely confined to the uppermost leaf (flag leaf) + of the analytes, whereas the last quadrupole monitored m/z 411 and 409 for reduced and normal HC-toxin respectively. Each transition was monitored with a dwell time of 150 ms and collision energy of 15 V, with ultrapure nitrogen used as the collision gas. Mass selection was achieved using the following ions: 439.3 for reduced HC-toxin and 437.3 for HC-toxin. Data were collected and analyzed via the MassHunter Workstation , and peak areas were determined by integration. Similar to Hm1A, the HCTR activity of the new APR alleles generated by targeted EMS mutagenesis (Hm1-3 and Hm1-4) along with resistant (Hm1) and susceptible (hm1-2) controls were also evaluated by LC-MS/MS.The solvent system contained solvents A (0.1% formic acid in ddHHm1Hm1hm2hm2) maize inbred, which exhibits complete resistance to CCR1 at all stages of plant development , and maize dihydroflavonol 4-reductase (DFR). Among the five amino acid substitutions present in HM1A (highlighted in red and bold), the L116 residue was a conserved in all Hm1 alleles and orthologs.Sequence comparison of HM1(PDF)Click here for additional data file.S3 Fighm1-2 is 176 amino acids similar to HM1-B73 and the mutation in the intron is predicted to generate 26 different amino acids before resulting in a truncated protein.(A) Ethyl methanesulfonate (EMS) mutagenesis caused a G to A substitution at the junction of exon3/intron3. (B) Predicted protein sequence of (PDF)Click here for additional data file.S4 FigHm1B73 plants were resistant at all stages of plant development. Two novel APR alleles, Hm1-3 (B) and Hm1-4 (C), generated by targeted EMS mutagenesis were susceptible as seedlings (week-3) and became resistant to CCR1 at week-7. A novel null allele, hm1-5 (D) remained susceptible throughout the age of the plant.(A) (PDF)Click here for additional data file.S5 FigHm1B73 allele over the hm1Pr allele from heterozygous M1 plants generated using the targeted EMS mutagenesis screen. Grey boxes represent the exons while the black lines between the exons represent the introns. Different primer pairs used to amplify Hm1B73 are marked using different colors with the amplicon size listed above each fragment.Four primer pairs that preferentially amplified overlapping sequences of the (PDF)Click here for additional data file.S1 TableUsing PCR-based markers, APR alleles were introgressed into B73 genetic background.(PDF)Click here for additional data file.


Objective: Uveitis is the most common extra-articular manifestation of ankylosing spondylitis (AS), for which no diagnostic biomarkers have been identified. This study was conducted to identify biomarker for uveitis in AS.Methods: To identify autoantibodies associated with uveitis in AS, we performed human protein microarray analysis using sera derived from various autoimmune diseases and ELISA analysis of sera derived from AS and rheumatoid arthritis patients. In the curdlan-induced SKG mice model, ophthalmic examination was performed at week 8 post-immunization and histologic examination of the ocular lesions performed at week 16 post-immunization. Serum levels of target antibodies were assessed at various time-points. To evaluate the functional role of specific autoantibodies, an in vitro apoptosis assay using ARPE-19 cells was performed.Results: Reactivity against prefoldin subunit 5 (PFDN5) was identified in AS with uveitis. Levels of anti-PFDN5 antibodies and PFDN5 in sera from AS with uveitis patients were significantly higher than those in AS without uveitis. At week 8, half of curdlan-treated SKG mice developed anterior uveitis, while all of them developed histologically confirmed uveitis at week 16. The levels of anti-PFDN5 antibodies increased over time in the sera of curdlan-treated SKG mice along with increased expression of PFDN5 and apoptosis in the ocular lesions. Knockdown of PFDN5 in ARPE19 cells resulted in increased apoptosis, suggesting a protective role of PFDN5 against cell death in uveitis.Conclusion: AS patients with uveitis have increased levels of anti-PFDN5 antibodies, and our findings suggest that anti-PFDN5 antibodies could provide a biomarker for uveitis in AS. Ankylosing spondylitis (AS) is an inflammatory disorder, characterized by both articular and extra-articular manifestations . Non-infSKG mice are suitable for establishing an animal model of chronic autoimmune arthritis . These mHere, we performed human protein microarray analysis of sera from patients with AS or other autoimmune diseases to identify biomarkers for uveitis in AS. We also conducted an animal study using curdlan-treated SKG mice to further evaluate the pathogenic role of the identified biomarker of uveitis in AS.ProtoArrays V4 (Invitrogen) were used to profile the autoantibodies present in Multiple Autoimmune Disease Genetics Consortium (MADGC) cohort sera. Arrays were scanned using a GenePix4000B Scanner and median pixel intensities were determined using GenePix Pro software version 5.0 (Axon Instruments). Sera from AS cohort at Hanyang university hospital  were collected and sera from age- and sex-matched healthy controls and rheumatoid arthritis (RA) patients were collected at the Asan Medical Center . This study was approved by the Institutional Review Board of Asan Medical Center, Seoul, South Korea (IRB No: 2015-0274) and Hanyang University hospital, Seoul, South Korea (IRB No: 2016-08-006).SKG mice with the BALB/c background were obtained from Dr. S. Sakaguchi  . Mice weSerum prefoldin subunit 5 (PFDN5) was measured with commercially available ELISA kit . Anti-PFDN5 was also measured by ELISA. Ninety-six well plates were coated with 1 μg/mL of recombinant human PFDN5 (Creative Biomart). Diluted serum (1:200) were added and were incubated for 2 h, followed by addition of secondary antibodies . Optical density was measured at 450 nm absorbance.At 16 weeks post-injection, whole-body sequential PET/MRI scanning was performed using a nanoScan PET/MRI (Mediso Ltd.). 18F-FDG (0.2 mCi/kg) was injected into the tail vein after a fasting period of at least 12 h, and a 30-min scan was initiated at 40 min after injection of the radioligand. After the MRI scan for 20 min, PET scan was performed for 10 min. MRI scans were acquired, and contiguous axial slices (1 mm) were obtained for the whole body. Scanning parameters were repetition time = 25 s, effective echo time = 3.4 ms, field of view = 64 mm, number of excitations = 1, frequency = 128, and phase = 128. Dynamic data acquisition of PET scans was performed 60–70 min after 18F-FDG injection. Acquired PET images were reconstructed using 3D full detector mode with MRI-based attenuation collection, with an energy level of 250–750 keV and 0.5-mm voxel size.in situ BrdU-Red DNA Fragmentation assay kit (abcam) was used according to the manufacturer's instructions. Stained slides were scanned with the multispectral Vectra scanner (Perkin Elmer).Sixteen weeks after curdlan injection , eyeball tissues from control and treated mice were fixed in 10% buffered formalin and paraffin-embedded. Sections (4 μm in thickness) were cut and stained with hematoxylin and eosin. For detection of apoptosis and necrotic cell death in eyeball tissues, ARPE-19, retinal pigment epithelial cells were maintained in DMEM/F12 (Thermo) with 10% FBS and 1% penicillin streptomycin. PFDN5 was silenced using small interfering RNA (siRNA). Cells were transfected with PFDN5 siRNA or scrambled RNA (Thermo) for 24 h using the RNA MAXi transfection reagent (Thermo) according to the manufacturer's instructions. Considering that endoplasmic reticulum (ER) stress is implicated in the pathogenesis of AS , we usedpost-hoc analysis) were assessed using Mann–Whitney U-test and t-test for non-normal distribution and non-normal distribution, respectively. Normality was assessed by Kolmogorov–Smirnov test. P < 0.05 were considered statistically significant.All analyses were performed using GraphPad Prism 5 software (GraphPad Software). Receiver operating characteristic curve analysis was performed to evaluate cut-off value of the particular biomarker and its area under the curve. Comparison among different groups were analyzed using Kruskal–Wallis test and ANOVA for non-normal distribution and normal distribution, respectively. Between-group differences (n = 21), juvenile idiopathic arthritis (n = 15), psoriatic arthritis (n = 34), pulmonary arterial hypertension (n = 34), AS without uveitis (n = 8), AS with uveitis (n = 8), we used high-density protein microarrays containing 8,087 human proteins. Increased antibodies to PFDN5 was detected in sera derived from AS patients with uveitis as compared to sera from patients with other diseases, including AS patients without uveitis (n = 25), AS without uveitis (n = 25), RA (n = 20), and healthy control (n = 10). Levels of anti-PFDN5 antibodies in AS patients with uveitis were higher than those in AS patients without uveitis (To investigate autoantibody reactivity in sera of patients with various autoimmune diseases in the US-based MADGC cohort consisting of RA ( uveitis . Interes uveitis . To conf uveitis . Conside uveitis .n = 6). Histologic exam was performed in 6 curdlan-treated SKG mice and 5 PBS-treated SKG mice at week 16 post-injection, because 4 curdlan-treated and 1 PBS-treated SKG mouse died before sacrifice. On H&E staining of orbital tissues, 6 of the 6 curdlan-treated SKG mice (100%) showed inflammation in ciliary body and in retina. In addition, damage in outer segment of photoreceptors extending to the inner layer of the retina was observed . Western blot revealed decreased expression of PFDN5 in ARPE19 cells transfected with PFDN5 siRNA (+Annexin V+ cells) in PFDN5 knockdown cells was significantly higher than that in control. Moreover, under tunicamycin stimulation, we observed a higher frequency of necrotic cells (7-AAD+Annexin V+ cells) among PFDN5 knockdown as compared to control cells  and anti-PFDN5 antibody  did not show any correlation with BASDAI. Thus, we suggest PFDN5 and anti-PFDN5 antibody as biomarkers of uveitis in AS rather than biomarkers of disease activity in AS.To assess the possibility of PFDN5 and anti-PFDN5 as a biomarker of disease activity in AS, we also analyzed the correlation of PFDN5 and anti-PFDN5 antibody with Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) in the Korean cohort (data not shown in the Results). Both PFDN5 (PFDN5 in mice causes retinal degeneration (We also observed a protective role of PFDN5 against apoptosis of retinal cells. PFDN is a hexameric protein that is exclusively found in archaea and eukaryotes , 12. It neration . Functioneration . In addineration . Our finneration . Based oIn our curdlan-treated SKG mice, histologic examination revealed inflammation as a pan-uveitis including both of anterior chamber and posterior segment. Although panuveitis may occur in some AS patients , typicalOur study has several limitations. First, we lack data regarding the severity of uveitis in the cohort patients at the time of sampling. These data, if were present might have provided the correlation between levels of anti-PFDN5 antibodies and severity of uveitis. Second, in the animal model, 5 curdlan-treated SKG mice had mild uveitis whereas none of the curdlan-treated SKG mice had moderate-to-severe uveitis on ophthalmologic exam at 8 weeks post-injection. Therefore, we were also unable to evaluate the correlation between levels of anti-PFDN5 antibodies and severity of uveitis in the animal model. Third, as non-AS uveitis patients were not included in both the US and Korean cohort, it is not clear whether anti-PFDN5 antibody is also specific to non-AS uveitis. However, our purpose was to identify biomarkers for uveitis, particularly in AS patients. For this purpose, we considered the difference in levels of anti-PFDN5 antibodies between AS patients with uveitis and AS patients without uveitis meaningful.In conclusion, we demonstrated increased levels of anti-PFDN5 autoantibodies in AS patients with uveitis. We also showed increased expression of PFDN5 in ocular lesions of curdlan-treated SKG mice, and demonstrate a protective role of PFDN5. Our data suggest anti-PFDN5 antibodies as a potential biomarker for diagnosis or prediction of uveitis in AS.WR and Y-GK designed the study. E-JL, WR, and Y-GK performed the experiments. OK, E-JL, JL, T-HK, SH, C-KL, BY, WR, and Y-GK analyzed the data. JY provided the SKG mice. OK, E-JL, WR, and Y-GK wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Virus-like particles (VLPs) derived from viral nucleocapsids are an important class of nanoparticles. The structure, uniformity, stability, and function of these VLPs have attracted scientists in utilizing them as a unique tool in various applications in biomedical fields. Their interaction with the innate immune system is of major importance for the adaptive immune response they induce. The innate immune cells and molecules recognize and interact with VLPs on the basis of two major characteristics: size and surface geometry. This review discusses the interaction of viral capsid-derived VLPs with the innate immune system. Virus-like particles (VLPs) are supramolecular complexes formed by viral proteins that self-assemble into capsids resembling real viruses, albeit non-infectious due to the lack of viral genome packaged within the particle. Such non-infectious particles can be naturally formed during infection or genetically engineered and produced in large scales in laboratories. Most VLPs are characterized by stability, together with uniform and repetitive structure that allow diverse applications in the field of biomedical science. Since their early discovery five decades ago, VLPs led to enormous progress in the field of vaccine development with seven vaccines approved for humans and several others in pre-clinical phase or in clinical trials ,2. Most The virus-like property of VLPs is a potent geometric pathogen-associated structural pattern (PASP) facilitating their engagement with innate and adaptive immune cells. The exterior and interior facets of VLPs can be functionalized and modified to enhance their immunogenicity and stability or to present heterologous antigens . The virsize and surface geometry [VLPs—especially those packaging nucleic acids—are capable of inducing a comparable humoral immune response to viruses. This is considered a major advantage of VLPs over attenuated virus-derived vaccines as it guarantees safety of the vaccines whilst retaining their immunogenicity . The immgeometry  as discuThe lymphatic system regulates the balance of fluid in the body as well as the trafficking of immune cells and antigens to lymphoid organs, in particular lymph nodes (LNs) . The effThe diameter of the blind-ended structure of the initial lymphatic vessels is about 10–60 μm. The vessels are lined up with single flattened endothelial cells forming valves that ensure the unidirectional movement of fluids through and along the lymphatic vessels . FurtherhiCD40hiCD8− and CD11cintCD40hiCD8− DCs—can successfully uptake VLPs after intradermal injection. Both identified subsets of skin-derived DCs have also been shown to effectively cross-prime cytotoxic T-cells following VLPs uptake [+ DCs are usually much more potent in cross-presenting VLP-derived antigens [The kinetics of free drainage of viral capsid-derived VLPs has been studied extensively and previous data has shown that VLPs with average size of 30 nm can be detected in mice footpads for at least 40 min post injection and at the draining LN 2 h post injection . Recentls uptake . Even thantigens .In summary, the size of antigen influences the immunogenicity largely by two mechanisms: first, by allowing the appropriate draining from periphery to secondary lymphoid organs and second by facilitating the interaction with APCs due to their repetitive surface. VLPs size distribution falls within the aforementioned optimal range, which explains in part its remarkable immunogenicity.In general, particles ranging between 20–200 nm upon reaching the LN are distributed throughout the different areas of the draining LN. Previous studies have shown that VLPs are detected at the sub-capsular, medullar and cortical regions of LNs as fast as 2 h post subcutaneous injection. At a later stage, VLPs are also detected deeper in B-cell follicles bound to follicular dendritic cells (fDCs) . At 48 h+F4/80+, different subsets of conventional dendritic cells (cDCs) including CD8+CD11c+, CD8−CD11b+, and CD8+CD11c+. B-cells characterized by CD45R/B220+ were less efficient in up taking VLPs [+ DCs for effective CTL priming [The predominant subsets of APCs actively up taking viral capsid-derived VLPs in the popliteal LN post subcutaneous injection have been recently classified. These cells include the sub-capsular sinus (SCS) macrophages CD11bing VLPs  but are ing VLPs . Medullaing VLPs . Therefo priming .As described above, the draining and trafficking of VLPs within the immune system and subsequent interaction with immune cells such as DCs, macrophages, and B cells allow the initiation of both humoral and cellular immune responses. Details of the immune response to VLPs used as vaccines will be discussed in the following sections.The highly repetitive surface structure of viral capsid-derived VLPs facilitates their interaction with components of the innate humoral immune system that in turn mediates opsonization and phagocytosis by APCs . The natThe pentraxin protein family including the short pentraxin C-reactive protein (CRP), serum amyloid P (SAP) and long pentraxin (PTX3) may also recognize the repetitive structure of VLPs due to high avidity interactions. SAP, for example, has been shown to bind to influenza A viral particles . This reThe hallmark of the immune response induced by VLPs is the induction of high levels of long lasting humoral responses. In contrast to soluble antigens, the highly organized and repetitive structure of VLPs promotes crosslinking of BCRs that surpasses the activation threshold and bypasses the initial need of T-cell help. As a consequence, any antigen exposed on the surface of VLPs in this same organized and repetitive array will benefit from this viral fingerprint and induce high levels of humoral responses.Another important feature of the humoral response elicited by VLPs is the promotion of isotype switching dependent on direct TLR7 or TLR9 signaling on B-cells ,28. UponFurthermore, intranasal administration of VLPs in murine models resulted in strong B-cell responses and germinal center formation in the spleen. Roaming B-cells in the lungs have been shown to bind VLPs through their BCR and shuttle them to B-cell follicles within the spleen .h CD4+ cells following endosomal processing [+ CTLs priming. The classical source of MHC-I antigens is newly synthesized proteins which are misfolded and degraded in the proteasomes. Nevertheless, it has been found that both presentation pathways are not strictly separated [h cells has been previously documented to be highly efficient and biologically relevant. Cross-presentation on MHC-I molecules has also been studied extensively and has been documented for tumor cells, necrotic cells, apoptotic cells, viruses, and VLPs [+ DCs only while CD8− DCs present them on MHC-II molecules [Following the uptake by professional APCs, processed VLPs will be subsequently presented on MHC molecules. In general, exogenous antigens (endocytosed or phagocytosed) are presented on MHC-II molecules to trigger the activation of Tocessing . Endogeneparated  as exogeeparated ,33,34. Ceparated ,36. The and VLPs ,38,39. Tolecules .+ and CD8− DCs can efficiently present VLP-derived antigens on MHC-II molecules, however only CD8+ DCs were able to cross-present VLPs derived peptides. This implies that VLPs presentation on MHC-II molecules to CD4+ T-cells is not restricted to a specific DCs subset but cross-presentation to CD8+ T-cells is usually restricted to a CD8+ DCs subset [However, this dichotomy hypothesis was not supported in other studies which have shown that both CD8s subset .h1 and CTL response [Many VLPs, including Norovirus, Papaya Mosaic Virus, Human Papilloma Virus and Qβ-derived VLPs activate innate cells through various means ,36,41. Aresponse . Non-metresponse . Class Aresponse . In contresponse . The homresponse . Free RNresponse . It has response .In general, recognition of ssRNA, dsRNAm or CpGs packaged into VLPs by endosomal TLRs will result in the activation of signaling pathways that are essential for the expression of different genes required to initiate the inflammatory responses . The proApart from TLRs, other PRRs and nucleic acid sensors have also been reported to play a role in the immune response elicited by VLPs. RIG-I and the stimulator of IFN genes protein (STING) induces significant levels of type-I IFN upon viral infection mediating autocrine and paracrine signaling . ActivatVLPs had been consistently successful at inducing protective immune responses in various pre-clinical models and clinical trials both for infectious and non-infectious diseases. The field had started employing naturally occurring VLPs during infection as vaccines, as exemplified by the Hepatitis B vaccine  and has The success of this approach relies on presenting heterologous antigens to the surface of model VLPs and in this manner, conferring the highly immunogenic viral fingerprint to those antigens. There are two main methods for presentation of heterologous antigens: chemical crosslinking or genetic fusion. The choice of method will impact the quality of the immune response as it will affect the valency of decoration of the particle with the antigen and the overall stability of the vaccine. For that reason, small changes in the crosslinking method must be closely evaluated as it can directly affect immunogenicity.A second important consideration for VLPs is the expression system of VLPs. During the assembly phase VLPs will randomly pack host derived components that can have an unbeknownst impact in the immune response. Thus, the same VLP can exert different immune responses depending on the manufacturing process. This is an important consideration when comparing results across different groups and when scaling up the manufacturing process.As is common to other vaccine development platforms, there are several challenges that should be considered for successful development of VLP-based vaccines. There are some risks that are specific to the VLP-platform technologies and some risks that are related to the specific vaccine candidates.Challenges specific to VLP-based vaccine platformsEven though several VLP-based vaccines are on the market, some more recent candidates struggle with stability. In addition, no vaccine that displays foreign epitopes has made it to the market so far. Hence, real-life, market PoC for such vaccines is missing. While there is no a priori reason that this should not be possible, it may still be perceived as a potential risk.Most if not all nucleocapsid VLPs derived from RNA viruses package RNA from the production host cells. This may need an additional Quality Control effort.If epitopes are to be fused into VLPs, this can create substantial problems, as VLPs may not assemble anymore.Challenges for individual vaccinesThe selected epitope may not be protectiveInduced immune responses may be too lowThe selected indication may sound interesting but does not attract interest from industry and/or the end-customerThe highly immunogenic properties of VLPs are a direct consequence of the viral fingerprint retained from the parental viruses. Such properties can be summarized by size, surface geometry, and ability to package nucleic acids. VLPs immunogenicity can also be extended by presenting heterologous antigens on their surface.


Through several mouse models, we demonstrated that TNF neutralization ameliorated ICB-exacerbated colitis in addition to improving ICB-dependent anti-tumor efficacy. Similar results were obtained after prophylactic TNF blockade in graft vs host xenografted mouse models with human immune cells, which showed a reduction in colitis and hepatitis. Importantly, there was a preservation of the immunotherapeutic control of xenografted tumors after ICB treatment. Moreover, TNF and TNF-dependent gene expression is upregulated in the colon mucosa from patients affected by colitis as a side effect of ipilimumab and nivolumab. Our results, thus, provide evidence of the successful combination of prophylactic TNF neutralization with ICB therapy strategy to ameliorate toxicities, while keeping or even ameliorating anti-tumor efficacy. The prophylactic TNF blockade strategy is clinically feasible since excellent TNF inhibitors have been approved for the treatment of autoimmunity and are used for the immune-related serious adverse events in immunotherapy.The TNF blockade therapy is currently a well-established treatment option for a variety of autoimmune diseases such as rheumatoid arthritis (RA), psoriasis or Crohn's disease, given the proinflammatory role of TNF in the course of these diseases. Importantly, TNF neutralization is also used for the treatment of corticosteroid-refractory immune-related adverse events (irAEs) induced by the combined anti-PD-1 and anti-CTLA-4 immunotherapy. The manifestation of these toxicities is an important limiting factor for the successful implementation of the inhibitory checkpoint blockade therapy (ICB), restraining its anti-tumor efficacy. In our recent study (Perez-Ruiz  Figure 1). The synergistic combination of PD-1 and CTLA-4 blockade therapy has been successfully used against a variety of tumors such as melanoma, renal cell carcinoma, or non-small-cell lung cancer with reportedly improved results when compared to single ICB therapy (C). Unfortunately, the appearance of serious immune-related adverse events (irAEs) has hindered the overall anti-tumor response of these therapies by forcing a reduction of the dose or, in over 30% of patients, discontinuation of the treatment . The course of action towards these irAEs consist of steroids treatment followed by transient or permanent treatment discontinuation and TNF blockade therapy is routinely used when there is no response to steroids. By using tumor-transplanted mouse models and xenograft mouse models, we have demonstrated that prophylactic blockade of TNF right before starting the dual anti-PD1 and anti-CTLA-4 therapy prevents irAEs, while maintaining or even enhancing to some extent the therapeutic efficacy of combined ICB therapy .Inhibitory checkpoint blockade (ICB) immunotherapy has produced very significant advances in the fight against cancer (The role of the TNF in the induction of ICB-mediated irAEs was proven in an autoimmune colitis mouse model that involved the induction of inflammatory bowel disease by dextran sulfate sodium (DSS). In this model, ICB therapy worsened the DSS-elicited autoimmune colitis with marked thickening of the intestinal wall and immune cell infiltration into the colon mucosa, signs that were prevented when TNF was neutralized by anti-TNF antibodies or etanercept. Importantly, the blockade of TNF resulted in an improvement of tumor survival in DSS-treated tumor-bearing mice after ICB therapy, unlike IL-6 neutralization, which harmed rather than improved ICB-enhanced anti-tumor responses. In the absence of DSS-induced colitis, ICB efficacy was retained when anti-TNF or etanercept was co-administered.+ T cells in the tumor microenvironment and tumor-draining lymph nodes (dLN) with the presence of gp70- and OVA-positive CD8+ T cells in MC38 and B16-OVA mouse models, respectively. Phenotypical analysis of intratumoral antigen-specific CD8+ T cells using a non-competing monoclonal antibody against PD-1 showed that these cells had a lower surface expression of this surface marker when mice received ICB therapy. Such downregulation of PD1 was also deadly observed when anti-TNF or etanercept was used. Interestingly, high levels of PD-1 have been correlated, along with other markers, with an exhaustion phenotype that it is characterized by functional impairment in CD8 T cells. These results raised the possibility that TNF-dependent ICB improved efficacy could be the result of a reduction in the proportion of antigen-specific CD8+ T cells that display an exhaustion phenotype. However, when the expression of various exhaustion markers  was analyzed, no differences were observed in any of them when compared to DSS-control treated antigen-specific CD8+ T cells independently of TNF neutralization. These data can be explained by a selective expansion of PD1-/low CD8+ T cells as a result of ICB treatment.The effectiveness of prophylactic TNF neutralization treatment in ICB therapy was linked to a higher abundance of infiltrated antigen-specific CD8+ T cells in the tumor microenvironment and dLN of mice as a result of systemic TNF neutralization, suggesting an attenuation of the activation-induced cell death (AICD). A similar reduction in apoptosis was obtained when T cell-receptor (TCR)-transgenic mouse CD8+ OT-I and Pmel-1 cells were activated in vitro with their corresponding peptides and co-cultured with anti-PD1/CTLA-4 in the presence of anti-TNF or etanercept in comparison with ICB treatment alone. Furthermore, the induction of AICD by the in vitro activation of human CD8+ T cells from healthy donors with anti-CD3/CD28 was reduced by infliximab or etanercept treatment. Thus, these data suggest that the mechanisms of action for TNF blockade seem to operate through a reduction of ICB-induced AICD. This immune phenomenon has been recently reported to limit the anti-tumor efficacy of the anti-PD-1/anti-CTLA-4 combined treatment in a low tumor burden setting characterized by tumor infiltration with partially exhausted T lymphocytes. In this report, IFN-γ was identified as a critical mediator of AICD, but IFN-γ blockade will likely affect the antitumor activity of T lymphocytes. Our data map the route of a clinically feasible strategy to promote the antitumor immune responses elicited by combined ICB, while preventing autoimmune reactionsAnother important difference observed was a reduction in the proportion of cell death within total and antigen-specific CD8TNF mRNA was significantly upregulated when compared to colon healthy tissue samples. In addition, a gene signature of TNF active tray was present in the mucosa of these patients. Using a xenograft-versus-host model disease, in which human peripheral blood mononuclear cells (PBMCs) were injected intravenously into Rag2-/-IL2rg-/- mice, we observed that ipilimumab and nivolumab treatment exacerbated immune-mediated colitis with an increase of weight loss and inflammation of the large intestine signified by the thickness of the intestinal wall. As expected from results obtained in mouse models, this effect was minimized by the co-treatment with etanercept. Moreover, in the presence of the human tumor cell line HT29 in the xenograft-versus-host model disease, etanercept treatment retained the ICB-mediated anti-tumor efficacy and decreased the level of alanine transaminase and aspartate transaminase in serum as well as reduced the thickness of the intestinal wall, all of which suggest a reduction in hepatitis and colitis that result from the xenograft from the host reaction. These results recapitulated the data obtained in mouse models and provide evidence for the potential benefits of using TNF blockade therapy to ameliorate the ICB therapy-dependent irAEs.To validate the results obtained in the mouse models and further probe the connection of TNF with ICB efficacy, we next performed an mRNA analysis of immune-related genes from biopsies of i) healthy mucosal tissue in cancer patients, ii) colon tissue from patients diagnosed with colitis after nivolumab and/or ipilimumab administration or iii) colon tissue from patients diagnosed with ulcerative colitis. In both ICB-mediated colitis and ulcerative colitis, Figure 1). Moreover, with this approach, a sustained and/or higher dose regimen of ICB compounds would be tolerated, which could strengthen its anticarcinogenic potential. Currently, a phase I investigator-initiated trial  is undergoing to evaluate the safety of the proposed approach. Sufficiently powered randomized clinical trials measuring overall response rate and the percentage of irAEs > grade 3 are the pathway forward.Despite the enormous efforts made to improve anti-tumor responses by ICB therapy, the manifestation of treatment-related toxicities has become a critical limiting factor for its applicability. Thus, efforts towards improving tolerability are required. Taking into consideration our findings, the administration of TNF neutralization agents prophylactically or concomitantly with ICB therapy was capable of resolving these shortcomings for ICB immunotherapy, while retaining its anti-tumor efficacy, presenting a strategy that allows for a separation between efficacy and toxicity (


Combinatorial VLPs vaccination showed significantly reduced inflammatory responses and lung viral loads upon rgH5N1 virus challenge infection, resulting in less body weight loss compared to M2e5x VLPs alone. These results indicate that immune responses to both M2e and HA might provide a strategy of vaccination inducing enhanced protection against avian influenza virus.Despite the ability to induce a broad range of cross protection, M2e5x virus-like particles (VLPs) alone provide limited vaccine efficacy and confer low efficacy of protection against highly pathogenic avian influenza virus (HPAIV) infection in chickens. Avian influenza hemagglutinin (HA) has been a major antigenic target that enhances humoral immunity, but the efficacy of avian HA-based vaccines against HPAIV needs to be further improved. In this study, we evaluated the vaccine efficacy induced by combination of conserved tandem repeat M2e5x VLPs and HA VLPs against an avian influenza virus. We found that combinatorial vaccine elicited higher levels of reassortant H5N1 (rgH5N1) virus-specific IgG, IgG1, IgG2a and IgA antibody responses compared to M2e5x VLPs in sera and lungs of mice. Combinatorial VLPs vaccination induced higher levels of CD8 Every year, highly pathogenic and contagious avian influenza causes thousands of poultry deaths, resulting in severe economic losses to the poultry industry . PoultryThe extracellular domain of ion channel M2 (M2e) is highly conserved among influenza A viruses and has been reported to be a target for cross protection. M2e5x virus-like particles (VLPs) have been generated by genetically engineering a tandem repeat comprising highly conserved M2e epitope sequences (M2e5x) from multiple host-origin influenza viruses with influenza matrix protein 1. M2e5x VLPs vaccine showed significant improvement in cross-protection in mouse models –5. M2e5xMost conventional avian influenza vaccines are based on the immunity to hemagglutinin (HA) protein. The hemagglutinin protein is a major target of protective antibody response induced by vaccination. HA-based vaccines inducing strain-specific immunity could be effective in the poultry industry. However, several factors limit vaccine efficacy, including variability and fast mutation rates of HA antigens of the virus. Thus, one of the major challenges in the poultry industry is in the development of broad-spectrum avian influenza vaccine, a similar problem against human influenza. Adenovirus-based influenza A virus vaccine containing hemagglutinin (HA) protein of the A/Vietnam/1203/2004 (H5N1) protected domestic chickens from an intranasal challenge with VN/1203/04 . VaccinaIn this study, we evaluated vaccine efficacy induced by combinatorial VLPs, which is a combination of two separate VLPs (one HA and another M2e5x), aiming to develop highly immunogenic vaccine. We found that combinatorial VLPs vaccination significantly reduced lung virus loads and inflammatory responses, providing better protection compared to M2e5x VLPs.Animal experiment in this study was carried out under the guidelines set out by Kyung Hee University IACUC. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Kyung Hee University (permit number: KHUASP(SE)-18-024). Immunization and bleeding were performed under mild anesthesia, which were induced and maintained with ketamine hydrochloride and xylazine. All efforts were made to minimize the number of animals used in the experiment as well as their suffering.Spodoptera frugiperda Sf9 insect cells were used for production of recombinant baculovirus (rBV) and VLPs in serum-free SF900 II medium . VLPs were generated by infecting Sf9 cells with recombinant baculovirus (rBV) at multiplicity of infection (MOI) of 0.05. [of 0.05. ,11. Madiof 0.05. . Inactivof 0.05. . Seven-wGGATCC ATGGA GAAAA TAGTG 3-; reverse primer, 5- ATAT AAGCTT TTAAA TGCAA ATTCT 3-; BamHI and HindⅢ sites are underlined). M1 gene from A/PR/8/34 was cloned as described previously[A/Viet Nam/1203/2004 (rgH5N1) virus was inoculated into MDCK cells and then total viral RNA was extracted using RNeasy Mini kit (Qiagen). Total RNA was reverse-transcribed to cDNA using Prime Script 1st strand cDNA synthesis kit  according to the manufacturer’s instructions. Avian influenza A/Viet Nam/1203/2004 (rgH5N1) HA gene was PCR-amplified with cDNA and primers containing restriction enzyme sites for cloning into the pFastBac plasmid expression vector , anti-M1 monoclonal antibody (GA2B)  and anti-M2e monoclonal antibody (14C2)  overnight at 4°C. HRP-conjugated goat anti-mouse IgG was used as secondary antibody. Additionally, M2e5x VLPs were also confirmed by enzyme-linked immunosorbent assay (ELISA). M2e5x VLPs were coated using various concentrations and confirmed by sequentially incubating with anti-M2 monoclonal antibody and HRP-conjugated goat anti-mouse IgG.2 pfu (50 μL PBS) of mouse lethal dose (5xLD50) of reassortant rgH5N1 virus containing HA and NA derived from avian influenza A/VietNam/1203/2004 (H5N1) and the remaining 6 internal genes from A/PR8 H1N1 virus. After monitoring body weight and survival, half of the mice were sedated and sacrificed 4 days post-infection (p.i.) for lung and spleen collection. Mice that displayed greater than 25% loss in body weight were considered to reach the endpoint of death and euthanized.Female, seven weeks old BALB/c mice were used. Groups of mice (n = 8) were intramuscularly (IM) vaccinated with HA VLPs (5ug) alone, combination VLPs (M2e5x VLPs 10μg + HA VLPs 5μg) or M2e5x VLPs (10μg) alone in 100μL at weeks 0 and 4. VLPs for immunization are suspended in PBS and naïve mice were given PBS only. The ratio of M2e5x with HA VLPs in combinatorial VLPs is 2:1, similar to previous studies ,15,16. AThe retro-orbital plexus puncture method was used to collect blood samples from mice at weeks 1 and 4 after prime and boost. To measure influenza-specific antibodies, flat bottom 96-well immunoplates  were coated overnight at 4°C with 100 μL of avian influenza A/Viet Nam/1203/2004 (rgH5N1) inactivated virus antigen at a final concentration of 4 μg/mL in 0.05M, pH 9.6 carbonate bicarbonate buffer per well. M2e-specific IgG antibody responses were also determined by using human influenza virus M2e peptide antigens as previously described . 100 μL in vitro culture.The viral titers in lung extracts were measured using MDCK cells as previously described. CytokinCells were prepared as indicated . Cell su+, CD8+) and B cell  from splenocytes and lung cells of mice were analyzed by flow cytometry 4 days post-challenge as described previously [6 cell/mL) in staining buffer  were incubated at 4°C for 15 min with Fc Block . For surface antigen staining, cells were incubated with fluorophore-conjugated antibodies  at 4°C for 30 min. Splenocytes and lung cells were washed with staining buffer and fixed with 4% paraformaldehyde for 30 min at 4°C before acquisition using a BD Accuri C6 Flow Cytometer (BD Biosciences). Data were analyzed using C6 Analysis software (BD Biosciences).The activity of immune cells in the spleen and lung was analyzed by flow cytometry as described previously . Percenteviously ,22. SpleAll parameters were recorded for individuals within all groups. Data sets were compared using non-parametric one-way analysis of variance (ANOVA) Kruskal–Wallis test and the ANOVA with multiple comparison test of PC-SAS 9.3 . A p-value < 0.05 was considered statistically significant.VLPs expressing HA or M2e VLPs with influenza M1 were confirmed by western blots and ELISA using HA, M1, and M2e specific antibodies. HA (62kDa) , M2e5x VThe levels of influenza-specific IgG, IgG1, and IgG2a antibody responses and M2e-specific IgG antibody responses in sera after prime and boost were measured. As shown in A goal of vaccination is to generate long-lived memory B cells that can rapidly differentiate into ASCs upon re-exposure to antigens ,19,23. T+ and CD8+ T and germinal center B cell populations were determined , virus titers in lungs were measured. Virus titer in Naive + Challenge was found to be significantly higher than vaccination group . The comHighly pathogenic avian influenza (HPAI) viruses are responsible for causing high mortality rates and substantial economic losses in poultry industry. However, reassortant influenza vaccines containing HA and NA genes from avian influenza virus cannot provide complete protection in chicken models . InactivM2e immunity induced by vaccination of mice with M2e5x VLPs have been reported to confer protection via antibody-mediated phagocytosis by macrophage and natural killer (NK) cell-dependent ADCC and HA-specific antibody prevents infection by neutralizing the virus . Thus, iMice immunized with either combinatorial VLPs or M2e5x VLPs alone significantly reduced both IFN-γ and IL-6 in lungs compared to Naïve + Challenge control, and the former of the two also displayed significantly lessened lung IFN-γ levels compared to the latter. A significant inverse correlation has been reported between the levels of inflammatory cytokines including IL-6 and IFN-γ and lung viral loads . In our + T cell and CD4+ T cell responses, whereas M2e5x VLPs only induced CD4+ T cell responses, indicating HA VLPs are more likely to contribute to inducing higher levels of CD8+ T cell responses. These findings were consistent with the previous study that reported vaccination with M2e5x VLPs induced low levels of M2e-specific cytokine producing CD8+ T cells, similar to those observed in naïve mice after infection [Interestingly, combinatorial VLPs showed high levels of CD8nfection .In summary, compared to M2e5x VLPs, combinatorial VLPs vaccination induced higher levels of H5 virus-specific IgG, IgG1, IgG2a and IgA antibody responses in sera or lungs and significantly reduced lung inflammatory responses, which may contribute to significant reduction of lung viral load and enhanced protection. These results indicated that combinatorial VLPs containing M2e5x VLPs and HA VLPs could be a better candidate in inducing improved vaccine efficacy against avian influenza infection.S1 Table(DOCX)Click here for additional data file.