extfieldsofstudy: Biology Medicine

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Histo-blood group antigen-binding specificities of human rotaviruses are associated with gastroenteritis but not with in vitro infection

Human strains of rotavirus A (RVAs) recognize fucosylated glycans belonging to histo-blood group antigens (HBGAs) through their spike protein VP8*. Lack of these ligands due to genetic polymorphisms is associated with resistance to gastroenteritis caused by P[8] genotype RVAs. With the aim to delineate the contribution of HBGAs in the process, we analyzed the glycan specificity of VP8* proteins from various P genotypes. Binding to saliva of VP8* from P[8] and P[4] genotypes required expression of both FUT2 and FUT3 enzymes, whilst binding of VP8* from the P[14] genotype required FUT2 and A enzymes. We further defined a glycan motif, GlcNAcβ3Galβ4GlcNAc, recognized by P[6] clinical strains. Conversion into Lewis antigens by the FUT3 enzyme impaired recognition, explaining their lower binding to saliva of Lewis positive phenotype. In addition, the presence of neutralizing antibodies was associated with the presence of the FUT2 wild type allele in sera from young healthy adults. Nonetheless, in vitro infection of transformed cell lines was independent of HBGAs expression, indicating that HBGAs are not human RV receptors. The match between results from saliva-based binding assays and the epidemiological data indicates that the polymorphism of human HBGAs controls susceptibility to RVAs, although the exact mechanism remains unclear.

. Biosynthesis pathway of HBGAs. HBGAs are synthesized by sequential addition of a monosaccharide to the terminal disaccharide of a precursor glycan. The synthesis pathways with type 1 and 2 precursors are considered here. Glycosidic linkages are separated by a forward slash to specify the pathway for different chains. The FUT2 and FUT3 fucosyltransferases drive the biosynthesis of secretor (Se) and Lewis (Le) antigens respectively by catalyzing the linkage-specific addition of α-fucose residues. Enzymes A and B catalyze the linkage-specific addition of N-acetylgalactosamine and galactose, respectively. Additions of glycans by enzymes A, B and FUT2 to the galactose residue of the precursor result in ABH HBGAs. Addition of α-fucose to the N-acetylglucosamine residue by FUT3 enzyme yields Lewis antigens (Lewis a/x, Lewis b/y, A Lewis b/y, B Lewis b/y).
Currently, two live-attenuated vaccines are licensed and available in many countries. Since their introduction, the number of deaths associated with RV infection has decreased from 450,000 to about 200,000 in 2011 32,33 .
The monovalent Rotarix ® vaccine (Glaxo-SmithKline) is derived from an attenuated human strain of G1P [8] genotype and the pentavalent RotaTeq ® vaccine (Merck) uses a bovine strain as genetic basis for five reassortants with human G1, G2, G3, G4 and P [8] 34 . According to recent studies, currently licensed vaccines are very effective (from 70 to more than 90% of protection) in high-income countries, but much less (20 to 30%) in low-income countries, which are the most affected 4 . Reasons for this lack of efficacy are not fully understood yet and probably multifactorial 35 . However, since both vaccines include a live-attenuated RV of P [8] genotype, the fact that Lewis negative children are resistant to P [8] strains could contribute to explain the reduced efficacy of vaccines in countries where the frequency of Lewis negative individuals is greater, such as Africa.
In light of these results, we make the hypothesis that RVA would first bind to fucosylated structures through their VP8* protein and secondly to sialic acids residues on gangliosides or to other cell surface receptors such as integrins or hsc70 14 , which would mediate virus entry. This hypothesis accounts for the fact that fucosylated structures are accessible in the glycocalyx thick layer, which length is around 500 nm on small intestine enterocytes, far more than gangliosides that extend no more than a few nanometers above the lipid bilayer 36 . With the aim to delineate the relative contribution of HBGAs in the infection process and to explore the consequences of HBGAs polymorphism on the virus transmission and efficacy of the available vaccines, we investigated the HBGA specificity of VP8* proteins from strains of various P genotypes, including the vaccine strains and the effect of HBGAs synthesis blockade on the in vitro infection of susceptible cells. We further analyzed the presence of neutralizing antibodies in sera from young healthy adults of known HBGA types.

P[8] and P[4] VP8* proteins bind to difucosylated structures.
We started by studying the binding of recombinant VP8* proteins from human strains to HBGAs by saliva-based binding assay since human saliva contains mucins that bear HBGAs, similar to those present at the surface of the small intestine 37 . VP8* proteins from vaccine strains (Rotarix P [8] and RotaTeq P [8]), cell culture-adapted strains (Wa P [8] and DS-1 P [4]) and three representative P [8] clinical strains (287, 183 and 134) were tested for binding to a panel of previously well-defined A, B, O, secretor and Lewis types saliva samples. The binding profile of VP8* proteins to HBGAs was identical for all the P [8] and P [4] strains tested, regardless of whether they were cell culture-adapted, vaccine or recent clinical strains (Fig. 2a). We observed a binding to mucins from secretor and Lewis positive (SE/Le + ) individuals significantly greater than the one observed with mucins of individuals either secretor but Lewis negative (SE/le − ), or non-secretor regardless of their Lewis phenotype (se/Le + and se/le − ) for which there was little or no binding at all (p < 0.001 or p < 0.01). Thus, the combination of SE and Le + phenotypes appears to be the minimum requirement for P [8] and P [4] VP8* proteins to bind to human salivary mucins. SE/Le + samples specifically contain the Lewis b difucosylated epitope (Fig. S1). Furthermore, direct binding of P [8] and P [4] VP8* to the difucosylated A Lewis b heptasaccharide was observed, whilst no binding occurred to related monofucosylated or non-fucosylated immobilized oligosaccharides, indicating requirement of the difucosylated Lewis b motif for binding (Fig. S2). Additionally, within the SE/Le + group, the ABO phenotype showed an effect with a significantly greater binding to O blood group mucins compared to B blood group mucins (p < 0.01 or p < 0.05) (Fig. 2b). Most P [8] VP8* proteins (Rotarix, RotaTeq, Wa, 287 and 134) showed an enhanced recognition of A blood group mucins as compared to B blood group mucins (p < 0.001 or p < 0.05). Two P [8] VP8* proteins (287 and 183) revealed a stronger binding to O blood group mucins than to A blood group mucins (p < 0.01 and p < 0.05). Binding of the DS-1 P [4] strain to the A blood group antigen was previously reported 21 . Yet, we failed to reveal a higher binding to saliva from the A blood group secretor phenotype as compared to O or B phenotypes. Consistently, no binding to the monofucosylated A blood group antigen by the P[4] DS-1 was observed using neoglyconjugates (Fig. S2). Moreover, saliva samples from the A SE/le − subtype were hardly recognized, unlike those from the A SE/Le + subtype, indicating that the difucosylated motif, not the A epitope by itself, is the dominant binding epitope for the DS-1 strain, similar to the P[8] strains (Fig. S3). Collectively, these results indicate that expression of difucosylated structures, requiring both FUT2 and FUT3 enzymes for their expression, is essential for efficient attachment of P [8] and P [4] VP8* proteins. The effect of the ABO phenotype, with a binding to saliva samples from blood group O ≥ A > B, suggests hindrance of the difucosylated ligand by addition of the galactose and for some strains of the N-acetylgalactosamine residues that constitute the B and A antigens respectively. P[6] VP8* proteins bind to Lewis negative saliva samples. Following P [8] and P [4], the P[6] genotype constitutes the third major P genotype of human RVs. Hence, we then investigated the binding of VP8* from P [6] clinical strains isolated in Burkina Faso to HBGAs by saliva-and synthetic oligosaccharide-based binding assays. We observed a preferential binding to Lewis negative saliva samples, irrespective of the secretor status (Fig. 3a). As this binding was not correlated with expression of the previously suggested H type 1 ligand (Fig. S4), we performed a synthetic oligosaccharide-based binding assay. This showed that P [6] VP8* proteins could bind to the Tk antigen, Le c 3′-LacNAc and GlcNAc3′-LacNAc6′-LacNAc (Fig. 3b,c and Table S1). These synthetic sugars have in common a GlcNAcβ3Galβ4GlcNAc-R carbohydrate sequence that likely constitutes the main recognition pattern of these strains (Fig. 4). Substitution of its terminal N-acetylglucosamine at position 4, giving either a type 2 precursor or the Lewis antigen, was not tolerated. Yet, a substitution at position 3 that gives the type 1 precursor by addition of a galactose residue was well tolerated. Branching of the glycan chain generated by addition of an N-acetylglucosamine residue in β6 linkage did not appear to influence the binding either. Overall, these data strongly suggest that precursor structures, including the extended type 1 precursor Galβ3GlcNAcβ3Galβ4GlcNAc-R constitute the ligand of P[6] VP8* proteins. This motif is available in the highest amounts on mucins from Lewis negative individuals since it is not masked by addition of the α1,4-linked fucose by the FUT3 enzyme. Nonetheless, precursor structures remain available in all individuals due to incomplete glycan synthesis 38,39 . This likely explains why the difference in binding between Lewis positive and Lewis negative individuals was not absolute.

P[14]
VP8* proteins recognize mucins expressing the A antigen whereas P[2] VP8* proteins do not bind to human salivary mucins. We then extended the study to the binding of VP8* proteins from two cell culture-adapted strains: a P [14] sialidase-insensitive humain strain (HAL1166) and a P [2] sialidase-sensitive animal strain (SA11). HAL1166 VP8* proteins bound to saliva samples from blood group A secretor individuals only (Fig. 5a) and to the A blood group antigen on noeglycoconjugates (Fig. S2). By contrast, SA11 VP8* proteins did not recognize human mucins (Fig. 5b). To control that this absence of binding was not due to a loss of functionality, we tested the binding of SA11 VP8* to MA-104 cells by flow cytometry. As expected, SA11 VP8* binding was readily detected on MA-104 cells and abrogated by sialidase treatment (Fig. S5). Thus, SA11 VP8* failed to recognize human salivary mucins.

Fucosylation is not a requirement for in vitro infection by P[8] and P[4] strains, unlike for infection of HT-29 cells by the P[14] HAL1166 strain.
In order to gain a better understanding of the contribution of HBGAs in the infection process, we then used an in vitro infection model. First, the expression of HBGAs on susceptible cells (HT-29, MA-104, Vero and Caco-2) was blocked using 2F-fucose, a metabolic inhibitor of fucosylation. Treatment efficacy was controlled by assessing expression of various HBGAs by flow cytometry. Control HT-29 cells, cultured in the presence of DMSO, express H, A and Lewis antigens. Expression of these structures on 2F-fucose-treated HT-29 cells was fully or substantially suppressed (Fig. 6a). Similar experiments conducted on MA-104, Vero and Caco-2 cells allowed us to draw the same conclusion, although initial phenotypes were different (Figs 7a, S6 and S7). Infection of these cells, either DMSO-(control) or 2F-fucose-treated, by several cell culture-adapted strains of RVs was then quantified by fluorescence microscopy. Infection by the sialic acid-dependent SA11 animal strain of P [2] genotype was used as control. It confirmed that fucosylation was not needed for infection by this strain since no significant decrease in infection was detected (Figs 6b, 7b and S6, S7). For the P [14] HAL1166 strain, there was a significant decrease in infection of 2F-fucose-treated HT-29 cells compared to control cells. Yet, that was not the case for the other strains (DS-1 P[4], Wa P [8], Rotarix P [8] and RotaTeq P [8] and P [5]) (Fig. 6b). Finally, no matter what strain was used, 2F-fucose treatment of other susceptible cell lines (MA-104, Vero and Caco-2) had no effect on infection (Figs 7b, S6 and S7). These data indicate that fucosylation and thereby expression of HBGAs is not a requirement for in vitro infection by P [8] and P [4] strains on transformed cells of non-small intestinal origin. However, in accordance with a previous study showing abrogation of virus infectivity in HT-29 cells by anti-A type antibodies 22 , our data indicate that fucosylation and more specifically synthesis of the A antigen is essential for in vitro infection by the P [14] HAL1166 strain. In agreement with the latter result, infection by the P [14] HAL1166 strain of Vero and Caco-2 cells that do not express the A antigen was very low. Intriguingly, infection by HAL1166 of MA-104 cells expressing the A antigen, like HT-29 cells, was not altered by its complete loss upon 2F-fucose treatment. Thus, the A antigen can be considered as virus receptor for P [14] strains on HT-29 cells, but not on MA-104 cells.
The presence of neutralizing antibodies is related to the presence of the wild type FUT2 allele.
Finally, we investigated the role of FUT2 genotypes in the neutralizing antibody response against the Wa human strain of P [8] genotype. Presence of neutralizing antibodies was determined by serum virus neutralization assay for 76 young healthy adults. Serum neutralizing antibodies as defined by a 50% inhibition above a 1/20 serum dilution, were present in 43 samples (57%), whereas no inhibition or inhibition below the threshold was observed for 33 samples (43%). The distribution of these two groups (with or without neutralizing antibodies) within the 3 FUT2 genotypes was then assessed. The number of individuals displaying neutralizing antibodies was significantly associated with the presence of the FUT2 wild type allele (p < 0.02) (Fig. 8). This observation indicates that the FUT2 genetic polymorphism plays a role in the susceptibility to RV infection that gives a long-lasting in vitro neutralizing antibody response. To confirm the binding profile to oligosaccharides, VP8* from another P [6] isolate (268) was also tested (c). Mann-Whitney test was used to compare groups (***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant).  20 showed that only children of the secretor geno-or phenotype, thus having at least one functional FUT2 allele, were found among those suffering from gastroenteritis caused by P [8] and P [4] RVs. Additionally, the study conducted in Burkina Faso, showed that no Secretor/Lewis negative child with gastroenteritis was infected by P [8] strains 26 . Moreover, the same study indicated that in case of infection by P [6] strains, Lewis negative children were at a greater risk of being among patients than Lewis positive children, regardless of their secretor status. These concordant data provide strong evidence that FUT2 (secretor) and FUT3 (Lewis) genetic polymorphisms contribute to susceptibility to major strains of RVs and therefore that HBGAs are involved in the infection process. Nonetheless, how and where in the body this takes place remains unclear.
In the present study, we produced VP8* proteins from clinical, vaccine and cell culture-adapted strains of several P genotypes, looking for relationships between their epidemiological profiles and their binding patterns to saliva samples of different HBGA types. There was no difference between the HBGA binding profiles of clinical and cell culture-adapted P [8] strains, indicating that HBGA binding was not a cell culture adaptation. We documented that all P [8] and P [4] VP8* proteins require both the secretor and Lewis fucosyltransferases (FUT2 and FUT3) for binding to saliva, indicating recognition of difucosylated structures. This is consistent with earlier results showing binding of P [8] and P [4] VP8* proteins to the Lewis b antigen 17 and to saliva samples from secretor individuals [18][19][20] . Interestingly, Huang et al. 17 observed a binding to the H type 1 antigen for P [8] and P [4] strains. However, saliva samples of secretor Lewis negative individuals (expressing the H type 1 antigen, but not the Lewis b antigen) were not recognized in our study. It is important to note that in the same study Huang et al. 17 observed specific binding of purified Wa triple layer particles to secretor Lewis b positive, but not to secretor Lewis b negative saliva samples, suggesting that binding to synthetic oligosaccharides may not perfectly mimic the natural ligands found on mucins. Regardless, the match between the results from the saliva binding assay and the epidemiological data suggests that the difucosylated Lewis b antigen may be a functional ligand in vivo for P [8] strains. P [8] and P [4] strains are the most commonly encountered worldwide and among the HBGA subgroups, the secretor and Lewis positive subgroup, which is preferentially recognized by P [8] and P [4] VP8*, is the most common (around 72-76% of the population in Europe). Behind P [8] and P [4], the P[6] genotype constitutes the third major P genotype of human RVs. Hence, we then investigated the binding of VP8* from two P [6] strains isolated in Burkina Faso. We observed that P[6] VP8* proteins bind preferentially to Lewis negative saliva samples, irrespective of the secretor status. This is consistent with findings of the epidemiological study conducted in Burkina Faso which showed that these same P [6] strains caused disease among Lewis negative children mainly, independent of their secretor status 26 , again illustrating the match between the saliva binding assay and epidemiological data. We observed that the glycan motif recognized by these clinical P [6] strains is a precursor structure comprised in the type 1 precursor Galβ3GlcNAcβ3Galβ4GlcNAc, although the terminal galactose does not appear to contribute much to the binding. Conversion into Lewis antigens by the FUT3 enzyme, as occurs in Lewis positive individuals, impaired recognition, explaining the lower binding to saliva of this phenotype. Our results are also compatible with those of previous studies showing binding to a group of long glycans containing the type 1 precursor HBGA terminal sequences, with or without the H epitope, but without the Lewis epitope 40,41 . Another previous study reported binding of the VP8* from a P [6] strain to A blood group oligosaccharide in solution 21 which contradicts the epidemiological data where a lower frequency of the A blood group was found among ill children infected by P [6] strains in comparison with the O blood group 26 . This discrepancy may be due to the use of an oligosaccharide in solution or of a cell culture-adapted strain. P [6] strains that have been most frequently found in individuals of the Lewis negative phenotype are seldom encountered in Europe where this phenotype represents only 5-10% of the population. Interestingly, its frequency can be as high as 30% in some Latino-American and African populations 42,43 where those strains are more common. Since mucins from Lewis positive individuals also contain the un-substituted type 1 precursor, albeit in lower amount than Lewis negative individuals 44 , Lewis positive children may also be infected, although less frequently, increasing to some extent the pool of susceptible individuals in the population.
We then extended the study to the binding of VP8* proteins from two cell culture-adapted strains: the HAL1166 sialidase-insensitive human strain of P [14] genotype and the SA11 sialidase-sensitive animal strain of P [2] genotype. We found that the HAL1166 VP8* proteins exclusively recognize mucins expressing the A antigen. This is in agreement with other studies showing that P [14] VP8* proteins recognize the type A HBGA 22,23 . As previously suggested by Liu et al. 26 , the fact that P [14] strains recognize the A antigen, which is a common carbohydrate shared between species, could explain why they are capable of causing disease to both human and other mammals. P [9], P [14] and P [25] strains exclusively recognize A blood group individuals who represent between 8-35% of the population depending on geographical areas 45 . Although, as yet, there is no epidemiological evidence indicating that only A blood group secretors are susceptible to disease due to these strains, it is likely that the limited frequency of this phenotype is too low to sustain transmission in humans, therefore explaining why they are seldom encountered. Collectively these observations point to a relationship between the frequency of genetically susceptible individuals in the population and the circulation of the viral strains.
VP8* from the SA11 sialidase-sensitive animal strain did not bind to human salivary mucins. N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the most common sialic acid family members with Neu5Gc differing from Neu5Ac by an additional hydroxyl group 46 . Interestingly, a previous study showed that Triple-Layered Particles (TLPs) from the SA11 strain strongly bind to the ganglioside   NeuGc-GM3, while they do not bind to NeuAc-GM3 47 . Therefore, the lack of binding to human saliva could be due to the absence of NeuGc, which is not expressed in normal human tissues but can be found in most mammalian tissues 48 . This could explain why P [2] strains can infect a wide variety of animal species, whilst the lack of binding to human saliva is associated with the lack of human infection 26 .
In addition to the glycan specificity of P [8] and P [4] strains VP8*, we also investigated the relationship between the RV-specific neutralizing antibodies in sera from healthy French blood donors and their FUT2 genotype. The presence of neutralizing antibodies is the result of previous infections and constitutes an indirect marker of virus susceptibility. Here, we showed that the probability of having neutralizing antibodies was associated with the presence of the FUT2 wild type allele. This is concordant with results of previous studies 20,29,30 suggesting that individuals with a secretor phenotype might be more susceptible to disease caused by RV infections as compared to non-secretors. Nonetheless, the fact that non-secretor individuals also exhibited neutralizing antibodies suggests that children with this HBGA phenotype might be somewhat susceptible to infection by P [4] or P [8] strains that constitute the vast majority of strains circulating in France 49 , or they might have been infected by strains of other P types that generated heterotypic antibodies. An alternative explanation for the observed relationship between the FUT2 polymorphism and the RV-specific neutralizing antibodies levels might stem from the impact of the FUT2 polymorphism on the microbiota composition that in turn would influence the antibody response to RV infection 50 . Several reports described associations between the FUT2 genotype and the gut microbiota composition 30,[51][52][53] . Yet, recent studies performed on large cohorts failed to confirm these associations 54,55 . Nonetheless, the microbiota composition of our cohort of 75 volunteers was determined. No significant association was found with the FUT2 genotypes, ruling out this possibility (to be published elsewhere).
The fact that the HBGA recognition is conserved between recent (clinical strains) and older (Wa and vaccine strains) P [8] strains suggests that HBGAs polymorphism may contribute to explain the modest efficacy of vaccines in areas where the frequency of FUT2 negative (non-secretor) and/or FUT3 negative (Lewis negative) individuals is high since these are areas where strains of other P types such as P [6] circulate most readily, as discussed above. It is also interesting to note that within the secretor/Lewis positive subgroup, we observed an effect of the ABO phenotype on binding to saliva samples of all P [8] strains. These results are in agreement with a previous study showing a lower binding of P[8] VP8* to blood type B saliva samples, suggesting also hindrance by addition of the galactose residue constituting the B antigen 17 . Interestingly, two recent studies conducted in Pakistan and Nicaragua, reported lower seroconversion rates after vaccination in nonsecretors as well as in non-blood group O secretors or B blood group secretors respectively 56,57 . The concordance of these new observations with the HBGA specificity of the vaccines VP8* strongly suggests that the FUT2 and ABO gene polymorphisms might impact the quality of immunization. Further epidemiological studies would be necessary to confirm these results and clarify whether the combinations of ABO, FUT2 and FUT3 subtypes and their variable frequencies among populations can contribute to explain the lower efficacy of RVA vaccines in some countries.
Since as described above, multiple studies point towards an essential role of HBGA-binding in the infection process, we studied the effect of fucose synthesis blockade on in vitro infection. That led us to conclude that fucosylation is essential for in vitro infection of HT-29 cells by the P [14] HAL1166 strain but it is not a requirement for infection of various cell lines by P [8] and P [4] strains. These findings suggest that HBGA-binding could be an early event which is not involved in the in vitro infection of poorly differentiated cells. Alternatively, the presence Figure 8. Relationship between the presence of neutralizing antibodies to the human RV strain Wa (P [8]) and the FUT2 genotype in French healthy young adults. Presence of neutralizing antibodies against the RV strain Wa (P [8]) was determined in sera from 76 healthy individuals by serum virus neutralization assay. The distribution of each genotype was as follows: 28 homozygous secretors (SE/SE); 29 heterozygous secretors (SE/ se) and 19 non-secretors (se/se). Titers were defined as the dilution of serum leading to a 50% inhibition of infection as compared to the positive control. Two groups of individuals were then defined: those for whom a titer was obtained and those for whom no titer was obtained. Chi-square test for trend was used to compare distributions (p < 0.02). of HBGAs might enhance viral replication and development of the disease but not be essential for infection in the strict sense. The use of more sophisticated cellular models such as Human Intestinal Enteroids (HIEs), will be necessary to clarify this point 58,59 .
In conclusion, our findings further indicate that human HBGAs expressed on host cells or digestive tissues may be used by the major P genotypes of circulating RVs as attachment factors or receptors. Alongside epidemiological data, this suggests that genetically controlled polymorphic human HBGAs affect the phylogeographic distribution of these viruses. Differential expression or frequency of HBGAs could also be of major importance in the host tropism and transmission of RVs between human and animal species. More experimental data are required to elucidate the mechanisms whereby HBGAs impact RV infection and disease. These data would be of primary interest for optimization of current vaccines or development of new vaccine strategies.

Methods
Production of VP8* proteins. Plasmids expressing the gene encoding the VP8* core proteins were synthesized by GeneART (Rotarix (P [8]), RotaTeq (P [8] strain) and SA11 (P[2])), cloned from cell culture-adapted virus (HAL1166 (P [14]), DS-1 (P [4]) and Wa (P [8])) or directly isolated from clinical stool samples (French isolates 134, 183, 287 (P [8]) and Burkinabe isolates 225 and 268 (P [6])) (see supplementary methods for GenBank accession numbers) as previously described 17 . In brief, complementary DNA produced from viral dsRNA by reverse transcription was used as template for PCR with specific primers to produce the VP8* gene fragment. VP8* gene fragments were cloned into the expression vector pGEX-4T-1 (GE Healthcare Life Sciences) between the SalI and BamHI sites. The final expression plasmid encoded for amino acids 46 to 224 of the VP4 protein fused to an N-terminal GST protein. After confirmation of the inserts through DNA sequencing, protein expression was performed mostly as described previously 17  VP8* binding to HBGAs. The binding of VP8* to HBGAs was measured by saliva-and synthetic oligosaccharide-based binding assays. A panel of previously well-defined A, B, O, secretor and Lewis types saliva samples was used as source of HBGAs (see supplementary methods for more information) in the saliva-based binding assays which were performed mostly as described previously 23 . Briefly, boiled saliva samples were diluted at 1:1,000 in 0.1 M carbonate-bicarbonate buffer pH 9.6 and coated onto 96-well microtiter plates (NUNC MaxiSorp) at 4 °C overnight. For the oligosaccharide-based binding assays, polyacrylamide neoglycoconjugates (IBCh, Russia), which are listed on Table S1, were coated at 20 μg/ml. After blocking with PBS containing 5% nonfat milk, GST-VP8* protein at 5 to 20 μg/ml was added and incubated at 4 °C overnight. The bound VP8* proteins were detected using a rat anti-serum to VP8* (see supplementary methods and Fig. S8 for more information) at 1:500 followed by HRP-conjugated goat anti-rat IgG (Sigma-Aldrich) at 1:5,000. In each step, the plates were incubated for 1 h at 37 °C and washed three times with PBS − 0.05% Tween 20. The signal intensities were displayed using a 3,3′,5,5′-Tetramethylbenzidine kit (BD OptEIA, BD Biosciences) and the absorbance was measured at 450 nm using a microplate absorbance reader (iMark, Bio-Rad). For the saliva-based binding assays, raw absorbance data are shown and background noise was comprised between 0.05 and 0.1. For the synthetic oligosaccharide-based binding assays, background noise was subtracted from raw data and was around 0.1. Saliva samples collection was approved by the Nantes University Hospital Review Board (study no. BRD02/2-P), informed consent was obtained from all saliva donors and all experiments were performed in accordance with the French national guidelines and regulations.

Infectivity assay. A description of cells and viruses used in this study is provided in supplementary meth-
ods. RV infectivity was activated with porcine pancreatic trypsin (Sigma) at 10 µg/ml for 1 h at 37 °C. Cells were washed twice with serum-free medium and inoculated with trypsin-activated virus at a multiplicity of infection (MOI) ranging between 0.5 and 5 depending on the cell type. Plates were then incubated at 37 °C for 45-90 min. After allowing virus attachment to cells, the inoculum was removed and serum-supplemented medium was added. The infection was left to proceed for 14-15 h at 37 °C in 95% (vol/vol) air with 5% (vol/vol) CO 2 . Infected cells in methanol-fixed cell monolayers were detected by staining cells using a goat polyclonal anti-RV serum (Bio-Rad Antibodies) and FITC-labeled rabbit anti-goat IgG (Fc) antibody (Bio-Rad Antibodies), both diluted at 1:400 in PBS containing 3% BSA. Cell nuclei were stained with DAPI. Plates reading was performed on an ArrayScan HCS (ThermoScientific) which gives percentages of infected cells after counting the total cell number (number of blue elements) and the infected-cell number (number of both blue and green elements).

Treatment of cells with 2F-fucose.
To inhibit the synthesis of fucosylated structures, a competitive inhibitor of fucosyltransferases, the 2F-Peracetyl-Fucose (Merck) was used. This inhibitor enters the cell by passive diffusion and is metabolized into a GDP-fucose analog (2F-fucose) to form an inhibitory substrate of fucosyltransferases 60 . Cells were seeded in 96-well plates or 25 cm² flasks and treated with 500 µM 2F-Peracetyl-Fucose (Merck) solubilized in DMSO in the adequate medium for 3 days, replacing the medium with fresh drug daily. Negative controls were performed by adding the DMSO vehicle only to the culture medium. Afterwards, treated cells were phenotyped by flow cytometry or infected by RVs. Flow cytometry phenotyping. MA-104, HT-29, Caco-2 and Vero cells from confluent cell monolayers were detached by brief treatment with trypsin-EDTA and resuspended in the appropriate culture medium containing 10% (vol/vol) fetal bovine serum. The following reactions were performed with viable cells (2.10 5 cells/ well of a 96-well plate) at 4 °C and incubation time was 30 min. The presence of histo-blood group H, A, B, Lewis a, Lewis b, Lewis x and Lewis y antigens at the cell surface was detected by indirect immunofluorescence staining (see supplementary methods for more information). Cell binding of GST-VP8* from SA11 was assayed as followed. GST-VP8* at 40 µg/ml was reacted with cells for 1 h. Cell-bound protein was detected with a rat anti-serum to VP8* diluted at 1:1,000. Similarly diluted normal rat serum served as a negative control. Bound rat antibodies were detected with FITC-conjugated goat anti-rat IgG (H + L) (Thermo Fisher Scientific) diluted at 1:200. After final washes in PBS containing 0.1% BSA and PBS only, flow-cytometry acquisition was performed on a BD Accuri C6 flow cytometer (BD Biosciences) and data were analysed using FlowJo software. The level of cell surface expression or GST-VP8* bound to cells was expressed as the mean fluorescence intensity (MFI). Serum samples. Serum samples used in this study come from the GOMMS PRL12009 project as a part of the biobank of Biofortis MérieuxNutriSciences. This biocollection is registered at the French Research Ministry (AC-2013-1792) and the PRL12009 project was approved by the French Ethic Comity (CPP Ouest IV). All volunteers were aware of the protocol of the study and fulfilled the informed consent form. They were aged 18-30 years and considered as healthy.
Serum virus neutralization assay. Sera from individuals of known ABO, secretor, and Lewis types (typed as previously described 24,61 ) were initially diluted at 1:20 using serum-free medium. Then, two-fold dilutions of all samples were made to reach a final dilution of 1:320. Simultaneously, infectivity of the human Wa strain was activated as described above. Serially diluted sera were pre-incubated with 2 × 10 3 FFU of the virus for 1.5 h at 37 °C before addition to MA-104 cells.
Statistical analysis. GraphPad Prism v 5.0 (GraphPad Software, San Diego, CA, USA) was used for data analysis. Comparisons for evaluation of VP8* proteins binding, cell infection and neutralization antibodies were performed by Mann-Whitney test, Wilcoxon signed-rank test, based on median values and Chi square test for trend, respectively. Differences were considered statistically significant when the level of two-tailed significance was p < 0.05. Data availability. All data analyzed during this study are included in this published article (and its Supplementary Information files).


Bio-Inspired Protein-Based Nanoformulations for Cancer Theranostics

Over the past decade, more interests have been aroused in engineering protein-based nanoformulations for cancer treatment. This excitement originates from the success of FDA approved Abraxane (Albumin-based paclitaxel nanoparticles) in 2005. The new generation of biocompatible endogenous protein-based nanoformulations is currently constructed through delivering cancer therapeutic and diagnostic agents simultaneously, as named potential theranostics. Protein nanoformulations are commonly incorporated with dyes, contrast agents, drug payloads or inorganic nanoclusters, serving as imaging-guided combinatorial cancer therapeutics. Employing the nature identity of proteins, the theranostics, escape the clearance by reticuloendothelial cells and have a long blood circulation time. The nanoscale sizet allows them to be penetrated deeply into tumor tissues. In addition, stimuli release and targeted molecules are incorporated to improve the delivery efficiency. The ongoing advancement of protein-based nanoformulations for cancer theranostics in recent 5 years is reviewed in this paper. Fine-designed nanoformulations based on albumin, ferritin, gelatin, and transferrin are highlighted from the literature. Finally, the current challenges are identified in translating protein-based nanoformulations from laboratory to clinical trials.

INTRODUCTION
Cancer, a leading cause of death globally, reaching approximately 8.2 million mortalities yearly, poses an enormous burden on society (Torre et al., 2015;Mehra et al., 2017). Cancer counts as a multifactorial and refractory disease caused by the local tissue micro-environmental and genetic factors intertwined (Hanahan and Weinberg, 2011). Common treatment strategies, including radiotherapy and/or chemotherapy with surgery, result in high treatment failure rate (Aoun et al., 2015;Kouchakzadeh and Abbas Shojaosadati, 2016). The reasons of treatment failure are generally diverse: (1) cancer is commonly detected at a later stage, thus accuracy and susceptibility of diagnosis and monitoring methods for early-stage cancer require further improvement (Ge and Liu, 2013;Torre et al., 2015); (2) in most cases, the conventional chemotherapy have been disappointing in efficacy due to multidrug resistance (MDR) and severe side effects (Gelperina et al., 2005;Pérez-Tomás, 2006;Szakács et al., 2006;Ge and Liu, 2013); (3) the cancer therapeutic biological agents (inclusive of antibodies, proteins and nucleic acids), the new class of anticancer drugs, are commonly unstable in in vivo circulation, with rapid degradation and inactivation before reaching the target site (Panyam and Labhasetwar, 2003;Sinha and Trehan, 2003). Accordingly, early detection, effective diagnosis and effective treatment of cancer are needed to be optimized to increase the survival rate and decrease the cancer associated deaths.
"Theranostics, " the portmanteau of therapeutics and diagnostics, has incorporated diagnostic and therapeutic functions into a single nanoplatform (Pene et al., 2009;Chen and Liu, 2016). It is noteworthy that the theranostics have been proposed as a new and revolutionary therapeutic concept in cancer therapy, enabling simultaneous diagnosis and treatment response monitoring using personalized medicine with high accuracy and specificity (Janib et al., 2010;Bardhan et al., 2011). Additionally, it is likely to incorporate numerous different therapeutic drugs into a single theranostics nanoplatform through judicious design to reach synergistic treatment of cancer Chen and Liu, 2016). In this regard, theranostics have become a research orientation arousing great concern and been promising in the field of cancer treatment.
This review article stresses the protein-based multifunctional theranostics progress in the past 5 years ( Table 1) and summaries critical foundation for further studying theranostics. The therapeutic methods include photothermal therapy (PTT), photodynamic therapy (PDT) and chemotherapy. The diagnostic or imaging methods included magnetic resonance (MR) imaging, fluorescence imaging, computed tomography (CT) imaging, photoacoustic (PA) imaging, positron emission tomography (PET) imaging and so on. The combinatorial strategies between diagnostic and therapeutic methods are summarized in Figure 1. Properties of commonly used proteins, i.e., albumin, ferritin, gelatin, and transferrin, were introduced. The preparation methods and key outcomes of in vitro or in vivo studies of these protein-based nanoformulations for cancer theranostics were elaborated.

Albumin and Its Properties
Albumins are obtained commercially in considerable amount from human serum (human serum albumin, HSA), bovine serum (bovine serum albumin, BSA), rat serum (RSA) and egg white (ovalbumin) (Karimi et al., 2016). Based on practicality, this review focus on HSA and BSA.
BSA shares ca. 76% sequence homology with HSA (Anand and Mukherjee, 2013), and is very soluble with a 69,323 Da molecular weight, consisting of 583 amino acid residues. BSA has an isoelectric point (pI) of 4.7 with a net charge of −18 mV (Anand and Mukherjee, 2013). BSA molecule is heart-shaped, consisting of three repeating domains (labeled I-III), with each of which falling into two sub-domains A and B (Majorek et al., 2012). BSA is extensively adopted for cancer theranostics by virtue of low cost, abundance, ease of purification and proper delivering properties.
HSA, the most abundant plasma protein (35-50 mg/mL human serum), is synthesized in the liver. HSA is a singlechain and non-glycosylated polypeptide with 66,500 Da in molecular weight, consisting of 585 amino acid residues, and heart-shaped with 80 × 80 × 30 Å in ca. dimensions (Sugio et al., 1999). From X-ray crystallographic analyses, the vital difference between BSA and HSA is that the former contains two tryptophan amino acid residues (Trp-135 and Trp-212), whereas the latter has merely one, Trp-214. HSA is very soluble, being extremely robust toward temperature (available at 60 • C for 10 h), pH (stable in pH 4-9) and organic solvents. Besides, HSA protein has preferentially been uptaken to tumor interstitium via the pathway of SPARC glycoprotein and gp60 glycoprotein transcytosis. These properties as well as its deficient toxicity, immunogenicity, and biodegradability make it an ideal candidate for cancer theranostics.

HSA for Cancer Theranostics
HSA has broadly served as a natural carrier to isolate organic molecules or inorganic oxide, inclusive of IR780, superparamagnetic iron oxide, IR825, and chlorin e6 (Ce6), for synthesizing effective theranostic agents.

Individual HSA-Dye Complexes
HSA is well-known with multiple hydrophobic binding pockets, and able to be bound non-covalently with many organic dyes, forming HSA-dye complexes with a high fluorescence quantum yield. In recent years, near-infrared (NIR) dyes, e.g., IR780, indocyanine green (ICG) and IR825, are broadly employed for cancer theranostics because of their relatively deep penetration and low interference. IR825 can be bound to the hydrophobic domain of HSA (the molar ratio of 1:1) via hydrophobic interactions, which fabricates a HSA-IR825 complex (Figure 2A) (Chen et al., 2014c). The HSA-IR825 complex have a high Frontiers in Pharmacology | www.frontiersin.org Transferrin Docetaxel-and quantum dots-loaded TPGS-Tf

Brain-targeted imaging and chemotherapy
The theranostic agent can effectively cross the blood-brain barrier and show fluorescence in the brain of rats. Sonali Singh et al., 2016 Transferrin Tf-IR780 NPs NIR imaging and PDT/PTT for Tfr-overexpressed tumors The CT26 tumor and liver can be clearly visualized at 2 h post-injection, while only the CT26 tumor can be clearly visualized at 12 h post-injection. After injection of the NPs followed by 808 nm laser irradiation, the CT26 tumor was effectively suppressed, and no significant adverse effect was observed.

Wang et al., 2016a
Transferrin Iron-dependent artesunate-loaded Tf-hollow mesoporous CuS NPs PA imaging and chemo-phototherapy for Tfr-overexpressed tumors The NPs can be effectively used for tumor imaging, and peritumoral injection is more conducive to tumor imaging than i.v. injection. After peritumoral injection of the NPs followed by 808 nm laser irradiation, the tumor was effectively suppressed.

Hou et al., 2017
Transferrin Holo-Tf-ICG Fluorescence and PA dual-modal imaging and PTT for glioma The theranostic agent can provide high spatial resolution fluorescence and PA imaging for visualization of the distribution of ICG in subcutaneous-and orthotopic-brain tumors. After i.v. injection of the theranostic agent followed by 808 nm laser irradiation, the U87 tumor was effectively suppressed, and no significant adverse effect was observed.
Frontiers in Pharmacology | www.frontiersin.org Oral administration of the theranostic agent exhibited significant antitumor efficacy in the human breast cancer tumor. Kanwar et al., 2016 fluorescence quantum yield at 600 nm excitation and an even high absorbance whereas low fluorescence quantum yield under 808 nm excitation, showing a great performance in NIR imaging and PTT at separated wavelengths. A gadolinium was further fabricated on HSA-IR825 for dual-modal imaging-guided PTT of tumor in a follow-up study ( Figure 2B) (Chen et al., 2014a). In this work, HSA was conjugated with the Gd(III) compound of diethylenetriamine pentaacetic acid, and further complexed with IR825, to form HAS-Gd-IR825 complex. The HAS-Gd-IR825 complex has great fluorescence and NIR absorbance, impressive T 1 relaxivity of 4.82 mM −1 s −1 . More recently, a gemcitabine functionalized HSA-IR780 agent was reported for chemotherapy and imaging-guided PTT of tumor (Han et al., 2017). HSA was first conjugated with gemcitabine via cathepsin B cleavable peptide GFLG, and then mixed with IR780 dye at the molar ratio of 1:1. IR780 could bound to HSA via hydrophobic interactions. For above systems, the NIR dyes were bound to the HSA via non-covalent interactions, possibly causing dye leakage during in vivo circulation (Rong et al., 2015). To address this problem, heptamethine CySCOOH dye (a NIR cyanine dye) was covalently conjugated to the lysine residues of HSA (Rong et al., 2015) via a modified EDC/NHS reaction for effective photoacoustic (PA), NIR fluorescence, thermal multimodality imaging and PTT. Such conjugation, compared with free CySCOOH dye under the identical conditions, resulted in higher PTT efficacy, tumor accumulation and longer circulation. Moreover, the maleimide group can be rapidly and selectively bound to the Cys34 residue of HSA via a Michael addition reaction. Recently, the Michael addition reaction was employed to couple fluorescent dye Cy7 to Cys34 position of HAS (Lisitskiy et al., 2017). And meantime, the chemotherapeutic agent pTFT (5-trifluoromethyl-2 ′ -deoxyuridine 5 ′ -monophosphate) was coupled to lysine residues of HSA via a redox and pH dual-sensitive linker. The conjugates could not only serve as an optical and 19 F MR imaging, but also be applied for delivery of chemotherapeutics.

HSA-Based Complexes
HSA has an effective diameter of 7.2 nm. Yet nanocarriers with sizes of 100-200 nm are well known to tend to accumulate in tumor tissues more efficiently via the enhanced permeability and retention (EPR) effect (Peer et al., 2007). Besides, theranostic agents usually require more functional ingredients to expand the application. Accordingly, great efforts have been devoted to design HSA-based complexes with appropriate sizes and more characteristics, to establish multifunctional HSA-based theranostics. For example, melanin and paclitaxel (PTX)-loaded HSA nanoparticles (HMP-NPs) with size of ca. 192 nm were fabricated with a desolvation-crosslinking method (Mo et al., 2007;Sim et al., 2015). The HMP-NPs showed effective PA signal intensity in the tumor site and the capability to tumor chemotherapy with long circulation time, as confirmed by in vivo experiments. In addition, the desolvation-crosslinking method was adopted to form HSA-based NPs (Pt(IV)-probe@HSA) for theranostic application (Li et al., 2015). In this work, the HSA NPs surface were conjugated with Pt(IV) antitumor prodrug, NIR fluorophore Cy5, and quencher Qsy21. The Pt(IV)-probe@HSA can not only selectively trigger the localized activation of Pt(IV) prodrug, but also enable real-time tumor cell imaging with high resolution.
For the above systems, glutaraldehyde is employed to stabilize the HSA NPs, whereas possible aldehyde residue may cause some side effects for in vivo applications (Fürst and Banerjee, 2005). To address this problem, some facile methods involving no toxic chemicals or exogenous cross-linkers have been broadly adopted in the preparation (Sheng et al., 2014;Hu et al., 2016). As an example, HSA-ICG NPs was developed with an average hydrodynamic diameter of nearly 75 nm based on the intermolecular disulfide bond cross-linking within HSA, for imaging-guided PDT and PTT treatments (Sheng et al., 2014). In this work, they first broke up the intramolecular disulfide bonds of HSA with the endogenous reducing agent glutathione, and then fabricated the HSA NPs with a desolvation method. A similar strategy was also adopted in their recent work to fabricate HSA nanoassemblies (NAs) with photosensitizer chlorin e6 (HSA-Ce6 NAs) for multi-modal imaging-guided PDT (Hu et al., 2016). The proposed HSA-Ce6 NAs had a diameter of ca. 100 nm, excellent tumor selectivity, promising triple-modal (fluorescence, PA and MR) imaging, and effective PDT properties.
Another method proposed by Liu's group not introducing exogenous cross-linkers is to fabricate HSA-based multifunctional theranostic NPs with drug-induced protein assembly strategy (Chen et al., 2015a). In this work, a multifunctional "Abraxane-like" theranostics agent was formulated through simply incorporating three clinical approved agents, ICG, PTX and HSA together ( Figure 2C). In this formulation, the NIR fluorescence imaging, thermal imaging, PTT and chemotherapy were efficiently combined. Accordingly, a synergistic therapeutic effect is demonstrated in treating metastatic and subcutaneous breast tumors. As a result, the strategy is enriched to design tumor-targeted theranostics agent for multimodal imaging-guided therapy of tumors (Chen et al., 2015b). HSA is pre-modified in their design, with either a tumortargeting acyclic Arg-Gly-Asp (RGD) peptide (HSA-RGD) or a photosensitizing agent Ce6 (HSA-Ce6). The anticancer drug PTX is then employed to induce the self-assembly of HSA-RGD and HSA-Ce6 to fall into two different NPs. After incorporation of manganese(II), both of NPs could be tracked by MR imaging and fluorescence imaging, which can be adopted for combinatorial cancer PDT and chemotherapy. Similarly, photosensitizer agent iron (II) phthalocyanine (FePc)-induced HSA self-assembly, is employed in a recent study to fabricate multifunctional HSA-FePc NPs for PA imaging-guided PTT . The as-prepared HSA-FePc NPs exhibited high stability, high PA imaging quality, efficient PTT treatment, and low long-term toxicity in vivo.

HSA-Coated Complexes
PTX-loaded HSA NPs were conjugated to the surface of porphyrin microbubbles for cancer theranostics . In this system, porphyrin microbubbles were fabricated using porphyrin-phospholipid conjugates to simultaneously intensify ultrasound and PA signal. The developed multifunctional theranostics agent is high sensitive in PA and ultrasound imaging, and effective in delivery of anticancer drug PTX to a tumor site. HSA-coated superparamagnetic iron oxide NPs were presented more recently, which can absorb pulsed microwave energy and transform efficiency into shockwave with the thermoelastic effect besides using as MR contrast agents (Wen et al., 2017).

BSA for Cancer Theranostics
Individual BSA-Dye Complexes BSA consists of multiple hydrophobic binding sites, and can naturally serve as a carrier of numerous small NIR dyes agents. Squaraine (SQ) was selectively bound to hydrophobic domain of BSA via hydrogen bonding and hydrophobic interactions with 80-fold enhanced fluorescence intensity . Based on this, a supramolecular adducts of SQ and BSA (SQ@BSA) was constructed and served as PTT agent and effective bioimaging probe simultaneously. In addition, folic acid (FA) functionalized SQ@BSA (SQ@BSA-FA) has additional functions, e.g., monitoring the time-dependent bio-distribution of adducts and targeting tumor sites. ICG, an FDA-approved NIR dye, can be adsorbed on BSA for NIR fluorescence imaging, PTT and PDT as excited by single-wavelength (Chen and Liu, 2016). Besides, BSA-based theranostics system covalently modified with NIR dyes has been reported . For example, a zinc-coordinated pH-sensitive theranostics agent is reported, consisting of two types of polyethylene glycol-BSA-imidazole covalently modified with either BHQ-3 quencher (NIR dark quencher) or Cy5.5 dye (donor NIR dye). At pH 5.0 (e.g., endo/lysosomes in cancer cells), the theranostics agent was disassembled rapidly, and emitted strong NIR fluorescence .

BSA-Based Complexes
Besides bound to NIR dyes, BSA can be further engineered with other functional agents via complexation, such as gadolinium (Wei et al., 2016;Yang et al., 2016;Chen et al., 2017a;Zhou et al., 2017), manganese Pan et al., 2017) and graphene's derivatives (Sheng et al., 2013). For example, hollow BSA was employed to fabricate a sizetunable Gd 2 O 3 @BSA conjugating Ce6 theranostics agent for MR imaging-guided PDT and PTT (Figure 3) . Notably, the BSA nanoreactor can not only effectively regulate the longitudinal relaxivity of Gd 2 O 3 , but conjugate readily with photosensitizers. A facile strategy is presented, adopting BSA as a biotemplate at physiological temperature, to construct a biocompatible Gd-integrated CuS multifunctional theranostics agent (Gd:CuS@BSA) . The fabricated Gd:CuS@BSA theranostics agent possessed ultrasmall sizes (about 9 nm), acceptable longitudinal r 1 relaxivity of 16.032 mM −1 ·s −1 , impressive temperature rise and intense PA signals under NIR irradiation. Recently, a multifunctional MoS 2 -Gd-BSA theranostics agent was fabricated through incorporating the good photothermal effect of MoS 2 nanoflakes with the high longitudinal proton relaxivity of BSA-Gd complex via the amine reaction between carboxyl groups of MoS 2 nanoflakes and amino groups of BSA-Gd . The MoS 2 -Gd-BSA theranostics agent exhibits a strong NIR absorbance and high r 1 relaxivity, which are helpful for PA and T 1 -weighted MR dual-modal imaging-guided PTT of cancer. Besides, a chemotherapeutic drug carmustine-encapsulated FITC-BSA NPs were prepared by desolvation/denaturation method and conjugated with a MR gadolinium(III) salt to form nanomedicine with dual imaging modalities (Wei et al., 2016).
At the identical stage, contrast agent Mn-based BSA complexes have been also reported for cancer theranostics Pan et al., 2017). For example, a multifunctional BSA-MnO 2 theranostics agent was fabricated via a mimicking drugsubstrate interaction strategy, adopting BSA as both reductant FIGURE 3 | Schematic illustration the synthesis of core-shell Gd 2 O 3 @BSA conjugating Ce6 (A) for MRI-guided cancer photothermal therapy (PTT) and photodynamic therapy (PDT) (B). Reproduced with permission . agent and template (Pan et al., 2017). It is noteworthy that ICG and PTX can be loaded on BSA-MnO 2 with a facile and energysaving mixing. The ICG/PTX-loaded BSA-MnO 2 theranostics agent could be adopted for MR imaging guided PTT and chemotherapy in vitro and in vivo.
Furthermore, a BSA-assisted synthesis route was developed to produce reduced graphene oxide (nano-rGO) theranostic agent (Sheng et al., 2013). The BSA functionalized nano-rGO displayed a high stability and low cytotoxicity, enabling PA imaging and PTT treatment without further surface modification. On that basis, a folate receptor active-targeted, pH-responsive BSA-pheophorbide-a (PheoA) photosensitizer incorporated GO nanocarrier (PheoA + GO:FA-BSA-c-PheoA NC) is developed as an theranostic agent (Battogtokh and Ko, 2016). The theranostic agent carries a good pH-responsive photosensitizer and generates a synergistic PTT and PDT effect against tumor by NIR irradiation.

BSA-Coated Complexes
BSA serves not only as a delivery platform to load various functional molecules, e.g., dyes and chemotherapy drugs, but also as a coating agent of other nanocarriers to increase the physiological stability, water-solubility and blood circulation time of theranostics systems. For example, BSA is employed to coat NaGdY 4 -based upconversion NPs (UCNPs), resulting in UCNP@BSA NPs possessing well stability and water-solubility in physiological environments . And meantime, two different dyes, consisting of an IR825 dye and a Rose Bengal (RB) photosensitizer, can be effectively loaded onto the BSA shell layer of the UCNP@BSA system. It is noteworthy that the characteristic absorbance peak of RB complies well with the green emission peak of UCNPs (980 nm excitation), which effectively kills cancer cells by PDT. Therefore, the dual-dye loaded theranostics agent can be adopted for MR diagnostic, upconversion optical imaging, PDT as well as PTT both in vitro and in vivo. BSA-coated magnetite Fe 3 O 4 core-shell structures with anticancer drug (gemcitabine) were developed, where BSA serving as the outer shell was further functionalized by the active targeted agent Anti-EGFR mab C225 . The theranostics agent can efficiently regulate doubletargeted thermochemotherapy against pancreatic tumor, monitor different cellular targeting by MR imaging, and distinguish various EGFR-expressing pancreatic tumor cells. On that basis, a BSA-coated magnetite Fe 5 C 2 theranostics agent was developed with a high loading of antitumor drug doxorubicin (DOX) (Yu et al., 2016). Notably, the DOX can be released in acidic condition and irradiated by NIR. In this regard, the theranostics agent serves as a smart nanoplatform for MR imaging, effective chemotherapy and PTT. A pH-responsive proteinpolymer bioconjugate-coated theranostics agent consisting of a superparamagnetic magnetite Fe 3 O 4 core, BSA-poly(ethylene glycol) (PEG) shell and anticancer drug DOX were presented for combined MR imaging diagnostics and chemotherapy (Semkina et al., 2015). A BSA-poly(ε-caprolactone) bioconjugate-coated upconversion theranostics agent is constructed with the similar preparation method for simultaneous cancer cell imaging, PDT, and chemotherapy (Dong et al., 2016). BSA stabilized Prussian blue (PB) NPs were fabricated, and ICG molecules were further attached non-covalently by a biocompatible and simple method (Sahu et al., 2016). Here, PB serves as a MR contrast enhancer. Accordingly, the multifunctional theranostics system could provide dual mode MR signal and NIR fluorescence imaging as well as combined therapy with PDT and PTT. A pH-responsive Au-BSA core/shell theranostics agent consisting of Au core, BSA shell that conjugated with DOX and FA was developed recently, which manifested tumor computed tomography (CT) imaging application and targeted cancer therapy (Huang et al., 2017).
Albumin-based NPs could be synthesized by using albumin as a scaffold, template, or stabilizer, and conjugating to polymers, drugs, and contrast agents. Covalent and non-covalent conjugation or assembly were employed. Due to the success of Abraxane, researchers preferred the non-convalent self-assembly. However, NPs prepared using the non-convalent strategy will vary batch to batch, which need to be overcome in the future studies .

Ferritin and Its Properties
Ferritin counts as an abundant protein in circulation, existing in intracellular and extracellular compartments. Ferritin is a 450 kDa hollow nanocage with internal and external dimensions of 8 and 12 nm, respectively (Banyard et al., 1978). It accumulates and stores approximately 4,500 iron atoms in a non-toxic whereas bioavailable form (Alkhateeb and Connor, 2013). In mammals, each ferritin protein consists of 24 subunits self-assembled into a spherical symmetrical protein shell (Alkhateeb and Connor, 2013). The ferritins from eukaryotes are produced by selfassembly of two subunit types, i.e., L-ferritin chain (19 kDa) and H-ferritin chain (21 kDa). The H-chain is centered by an iron oxidase center, required to oxidize Fe(II) to Fe(III), whereas the L-chain without ferroxidase activity nucleates iron (Bellini et al., 2014). Ferritin counts as a multifunctional protein with iron storage and metabolism. Ferritin is critical for angiogenesis, proliferation and immunosuppression, as demonstrated by growing number of evidence (Alkhateeb and Connor, 2013).
Ferritin is a ubiquitous protein robust extremely: it can be reversibly disassembled in the extremely acidic pH (pH 2-3) or basic pH (pH 10-12) and can be heated at 80 • C exerting no deleterious effects (Truffi et al., 2016). These properties as well as its biocompatibility and biodegradability make it a suitable candidate for cancer theranostics (Bhushan et al., 2014).

Ferritin for Cancer Theranostics
The ferritin not only provides a reaction vessel to fabricate numerous non-native metallic NPs inside its core, but also serves as a nanocarrier for various applications (Bhushan et al., 2014). For example, Zn hexadecafluorophthalocyanine (ZnF 16 Pc), a potent hydrophobic photosensitizer, is well encapsulated into the Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys (RGD4C)-modified ferritins (P-RFRTs) with a loading rate as high as ∼60 wt % for effective PDT (Zhen et al., 2013b). The P-RFRTs were further conjugated with ZW800 (a NIR dye molecule) in this work to better track P-RFRTs particles. With the identical approach, the DOX-loaded RGD4C-modified ferritin was also reported (Zhen et al., 2013a). A self-assembly encapsulation strategy based on step-wise change of pH was developed in another report to fabricate ferritin NPs with NIR dye IR820 for fluorescence/PA multimodal imaging-guided PTT . The IR820-loaded ferritin nanocages can effectively treat and diagnose cancer adopting two different excitation wavelengths, i.e., 550 nm for high quantum-yield fluorescence imaging, and 808 nm for PA imaging and effective PTT. Additionally, CuS was fabricated inside the cavity of ferritin nanocages with a biomimetic and straightforward synthesis strategy (Wang et al., 2016b). The CuS-ferritin has strong NIR absorbance, high photothermal conversion efficiency, good biocompatibility and distinct PA contrast,. Notably, the 64 CuSferritin theranostic system, as incorporated with radionuclide 64 Cu, also served as an excellent PET imaging agent.
The higher level of L-ferritin in tumor versus normal tissue has been observed in some malignancies tissues like breast cancer, colon cancer, pancreatic cancer and testicular seminoma (Alkhateeb and Connor, 2013). High amount of L-ferritin is usually associated and bound with intensified expression of the L-ferritin receptor that mediates ferritin endocytosis. The higher the expression of L-ferritin receptors, the more intensified uptake of L-ferritin in breast cancer MCF-7 cells (Geninatti Crich et al., 2015). Based on this, a ferritin-based nanotheranostic system has been further developed to simultaneously deliver a MR contrast agent GdHPDO3A and a natural anticancer molecule curcumin (Geninatti Crich et al., 2015). The theranostics system selectively delivered therapeutic and imaging agents to breast cancer cells. In a follow-up study, L-ferritin was found to target breast cancer stem cells (Conti et al., 2016). In this regard, ferritin was further exploited to deliver curcumin and the GdHPDO3A MR contrast agent simultaneously for breast cancer stem cells (Conti et al., 2016). In a very recent report (Turino et al., 2017), multifunctional theranostics system was developed through coating poly (lactic-co-glycolic acid) NPs (PLGA NPs) with L-ferritin to increase their targeting capability to breast cancer MCF-7 cells. The L-ferritin functionalized PLGA NPs loaded with an amphiphilic Gd based MR contrast agent and PTX for MR imaging guided chemotherapy. In addition to this, ferritin coating makes PLGA NPs more stable, thus avoiding the non-specific and fast release of therapy/diagnosis agents before reaching the targeted sites.
Hollow structure make the ferritin become an ideal carrier, however, after loading therapeutic agents, the slight change in conformation and activity might take place. The toxicity of ferritin-based NPs should be studied carefully as other NPs. Also, the surface-modified ferritins may be recognized as foreign substances. Therefore, when surface targeting ligands were needed, the grafting density are important factors to be considered.

Gelatin and Its Properties
Gelatin is a polyampholyte protein having both anion and cation along with hydrophobic groups (Elzoghby, 2013). Gelatins have repeating sequences of proline, alanine, and glycine amino acid triplets, which are essential for gelatin (Sahoo et al., 2015). They are obtained from alkaline-, acid-or enzymatic-hydrolysis of collagen. These chemically treated gelatins have varying isoelectric points, i.e., 4.5-6.0 for alkaline treatment and 7-9 for acid treatment (Patel et al., 2008;Ninan et al., 2011). In addition, anticancer agent release profiles from gelatin protein can be controlled through changing the molecular weight, gelatin source and the crosslinking degree (Foox and Zilberman, 2015). Gelatin is natural, biocompatible, biodegradable, water permeable, nontoxic, and soluble in water (Nezhadi et al., 2009). As a delivery carrier of therapeutic/diagnostic agents, gelatin has been revealed multifunctional properties, enabling the development and design of versatile theranostics.

Gelatin for Cancer Theranostics
Gelatin has native hydrophobic and hydrophilic segments in each single polypeptide chain, enabling itself a conducive coating agent for various metal NPs (Li et al., 2013;Cheng et al., 2014;Tran et al., 2017b). For example, a theranostic system was developed through coating iron oxide NPs with selfassembled gelatin (abbreviated as AGIO) (Li et al., 2013). In the meantime, calcium phosphate (CaP) and anticancer DOX could be loaded on gelatin by electrolytic co-deposition technique. The fabricated AGIO@CaP-DOX NPs had efficient MR contrast, superior cytocompatibility and potent cellular internalization toward HeLa cells. A gelatin coated multifunctional nanosystem, with oleylamine-coated Fe 3 O 4 NPs core, amphiphilic gelatin shell, and fluorescent labeling molecules FITC and antitumor platinum(IV) prodrug, was elaborated for fluorescence and MR imaging guided chemotherapy (Cheng et al., 2014). Most recently, oleic acid and gelatin were adopted to modify a silicacoated iron oxide magnetic NPs, which was demonstrated to encrease biocompatibility and solubility of iron oxide magnetic NPs and facilitate treatment-response monitoring of the tumors (Tran et al., 2017b). Particularly, the oleic acid and gelatin coated NPs enables the hydrophobic anticancer drug PTX to be loaded into the lipophilic oleic acid-gelatin shell. The synthesized theranostic system have high r 2 value, low cellular toxicity, high drug delivery efficacy and well anticancer efficacy in vitro. The pharmacokinetics, bio-distribution, tumor diagnostic and antitumor efficacy of the theranostic system in vivo were studied in a follow-up study to further confirm its potential in clinical applications (Tran et al., 2017a).
Beside iron oxide NPs, gold NPs are commonly coated with gelatin to increase their stability and biocompatibility. For example, a gelatin-coated gold NP-based nanostructure was developed for fluorescence imaging-guided chemotherapy (Tsai et al., 2016). The gelatin was firstly covalent conjugated with DOX, and then coated onto the epigallocatechin gallate (EGCG)-functionalized Au NPs. Here, DOX serves both as an anticancer drug and a fluorescent indicator. The attained DOX-gelatin/EGCG Au NPs exert an apparent inhibitory effect on the proliferation of human prostate cancer cells (PC-3) and can trace the intracellular enzyme-induced release of DOX through measuring the recovery of the DOX fluorescence signal. The covalent conjugation of DOX to nanocarriers may cause problems, e.g., insufficient in vivo release and decreased drug activity (Suarasan et al., 2016). A new temperatureand pH-responsive theranostic system was developed on the basis of DOX non-covalently bound to biosynthesized gelatin-coated Au NPs (DOX-AuNPs@gelatin) (Suarasan et al., 2016). The fabricated DOX-AuNPs@gelatin would be an ideal agent for cancer theranostics based on its good biocompatibility, high DOX loading capacity via non-covalent complexation and effective DOX release under the tumor environment.
Recently, angiopep-2 modified gelatin-based core-shell NPs (Angio-DOX-DGL-Gel-NP) have been designed to increase the tumor targeting efficiency, tumor retention and tumor penetration (Hu et al., 2015). In this system, the shell consisted of dendrigraft poly-lysine linked with angiopep-2 and DOX, whereas the core was made up of gelatin NPs degraded by matrix metalloproteinase-2. Fluorescence imaging-guided chemotherapy showed enhanced antitumor effects in vitro and in vivo, Gelatins are used as carriers in versatile drug delivery systems, form nanoparticles to microparticles (Foox and Zilberman, 2015). Small drug molecules or large bioactive molecules were easily entrapped into gelatins and released in a controlled manner. However, the cancer theranostic platforms based on gelatin didn't attract much attention. In our opinion, the gelatin-based NPs are promise cancer theranostics, especially in oral administration and brain delivery.

Transferrin and Its Properties
Transferrin is a monomeric glycoprotein with 679 amino acids and a large molecular weight of approximately 79 kDa (Parkkinen et al., 2002;Gomme et al., 2005). The molecule is protected by 3 carbohydrate side chains, one of which is O-linked (Ser-32) and the other are N-linked (Asn-413 and Asn-611) (Gomme et al., 2005). The polypeptide chain falls into two structurally similar lobes, known as the C-lobe (343 amino acids) and Nlobe (336 amino acids), which are connected by a short linear spacer sequence (Brandsma et al., 2011). Each lobe consists of one reversibly binding site for ferric iron with nearly 10 22 M −1 affinity at pH 7.4 (Aisen et al., 1978). The major biological function of transferrin is to distribute and control circulating Fe, which is required for numerous biological processes, including cellular metabolism and proliferation, DNA synthesis, electron transfer, and oxygen transport (Dufès et al., 2013;Tortorella and Karagiannis, 2014). Particularly, transferrin specifically binds transferrin receptors (TfRs) on cell surface, forming a transferrin-TfR complex, and gets internalized by receptormediated endocytosis (Szoke and Panteghini, 2012;Dufès et al., 2013). Meantime, overexpression of TfRs has been observed in various cancer cells, including breast carcinoma, glioblastoma, melanoma, ovarian carcinoma and colon carcinoma (Calzolari et al., 2007;Tros de Ilarduya and Düzgüneş, 2013).

Transferrin for Cancer Theranostics
As a promising tumor targeting ligand, transferrin is frequently adopted to facilitate targeting delivery of theranostics. For example, transferrin-functionalized graphene quantum dots were developed to track and image tumor cells expressing the TfRs . In the meantime, the anticancer drug, DOX, was adsorbed on graphene surface in the system via hydrophobic interactions and π-π stacking, which could be used for fluorescence imaging-guided chemotherapy. Another transferrin-conjugated PEGylated fluorescent nanodiamond with Dox payload was developed, and its targeting ability and chemotherapeutic potential were investigated in the human hepatoma (HepG2) cell lines with overexpression of TfRs and normal cell lines (L-02) with low-expression of TfR, respectively . A transferrin conjugated theranostic micelles of D-alpha-tocopheryl PEG 1000 succinate (TPGS) were synthesized, containing both ultra-bright gold clusters as a model imaging agent and docetaxel as anticancer drug for synchronous cancer imaging and therapy (Muthu et al., 2015). The transferrin conjugated micelles, compared with the nontransferrin functionalized theranostic micelles, showed higher cellular uptake, higher cytotoxicity in MDA-MB-231-luc breast cancer cells. In a follow-up study, a transferrin decorated TPGS coated theranostic liposomes was further developed for targeted co-delivery of quantum dots and docetaxel for imaging-guided chemotherapy of brain cancer (Sonali Singh et al., 2016). Indeed, the transferrin decorated theranostic liposomes, compared with the non-transferrin targeted preparations, showed an improved and prolonged brain targeting of quantum dots and docetaxel. In addition, rattle-type theranostic NPs of mesoporous silica-coated Fe 3 O 4 were develop by conjugating transferrin for targeted codelivery of NIR dye (Cy7) and hydrophobic anticancer drug PTX for NIR/MR bimodal imaging guided chemotherapy (Jiao et al., 2015).
Recently, transferrin and nuclear-targeted TAT peptide (YGRKKRRQRRR) conjugated magnetic NPs were proposed for PTT application (Peng et al., 2017). Magnetic NPs is preconjugated with transferrin and TAT peptide, and then bound to NIR dye Cy7. The theranostic system can efficiently target cancer cell nucleus and facilitate the NIR and MR imagingguided PTT. A core-shell theranostic system UCNP core (NaYF 4 :Gd 3+ , Yb 3+ , Er 3+ ) was developed, taking advantage of efficient NIR-to-visible up-conversion capability and increased tumor targeting ability and biocompatibility . In the meantime, a clinically approved PTT agent, protoporphyrin IX, was loaded into the transferrin shell, being able to be drawn upon by cancer cells for efficient PDT with NIR irradiation and luminescence bio-imaging. A new strategy (diffusion molecular retention tumor targeting effect) was developed to fabricate tumor-targeted theranostic system for PA imaging-guided chemotherapy/photothermal with synergistic effect (Hou et al., 2017). In this study, hollow mesoporous CuS NPs (HMCuS NPs) were conjugated with transferrin and iron-dependent artesunate (AS), an effective anticancer drug. In this regard, the attained AS/Transferrin-HMCuS NPs facilitated local drug accumulation and retention, targeted to breast cancer MCF-7 cells specially via TfR-mediated endocytosis, combined chemotherapyphototherapy synergistically, and eventually improved the anticancer effect.
Transferrin is not only a conducive targeting ligand, but also able to load imaging and/or therapeutic agents. For example, a simple, effective and safe self-assembly strategy was developed to fabricate transferrin NPs with NIR dye IR780 (Transferrin-IR780 NPs) for targeted imaging and phototherapy of cancer . The fabricated Transferrin-IR780 NPs had advantages on potent photo-stability, narrow size distribution as well as prominent photothermal conversion efficiency, and exhibited pronounced targeting and theranostics potential. A conjugate of transferrin, NIR dye Cy5.5 and cytotoxic chelating agent (NNE3TA:2,2 ′ -(7-(2-((carboxymethyl)(4nitrobenzyl)amino)ethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid) (NNE3TA-Transferrin-Cy5.5) was developed to treat and detect cancers (Kang et al., 2016). Targeted iron chelation cancer therapy and NIR imaging were demonstrated effectively in vitro with NNE3TA-Transferrin-Cy5.5. Most recently, a drug-induced transferrin self-assembly strategy is developed to fabricate tumor-targeted NPs for fluorescence and PA dual-modal imaging-guided PTT of glioma (Figure 4) (Zhu et al., 2017). In this system, transferrin could effectively load ICG via hydrophobic interaction and hydrogen bonding. Specifically, the preparation method is safe, simple and mild without the use of any toxic reagents. The obtained Transferrin-ICG NAs showed effective active tumortargeting, good biocompability, prominent dual-modal imaging as well as PTT efficacy, and could be adopted for theranostics of both subcutaneous and orthotopic brain tumors.
Notably, transferrin was employed not only as an delivery vehicle but also as a targeting agent in these works. It has been reported than transferrin-functionalized silica NPs lost targeting abilities completely in serum-rich media, due to shielding effects of the adsorbed proteins (Salvati et al., 2013). The bioavailability of the large transferrin determines the targeting functionality in complex biological media. It is a challenge to maintain the targeting efficiency of transferrin-based NPs in vivo.
Although natural proteins were used in most studies, the disadvantages of these protein-based platforms were obvious, such as poor permeability, ease of degradation, and potential immune responses. Moreover, after functionalization or conjugation of other molecules, the protein properties and functions might be altered. In recent years, encapsulation of proteins by in situ polymerization was developed as an alternative strategies for protein modification (Ye et al., 2016b). The protein nanogels were obtained by incorporation of crosslinkers to protein surface and polymerization. Such protein nanogels can maintain the protein properties and functions in complex chemical or biological environment, and avoid the immunogenicity of proteins. The out layer polymers were cleavable and degradable, while the inner proteins could be released by internal and external stimuli. Numerous therapeutic proteins have been developed using this method for cancer therapy (Ye et al., 2016a(Ye et al., , 2017.

CONCLUSION AND PROSPECTS
Natural proteins as biocompatible nanocarriers are broadly adopted in delivering therapeutic and diagnostic agents simultaneously. These nanoformulations are fabricated progressively complex and "smart" to employ multi functions in one platform. To increase the accuracy of diagnostic and the efficiency of cancer therapy, "all-in-one" nanoplatforms are designed with more than two imaging strategies and more than two therapeutic methods. Surface modification with targeted molecules and controlled drug release are employed to spatially control the localization of administered NPs. The improved efficiencies in tumor imaging and ablation are verified both in vitro and in vivo. Despite these huge advances, the translation of natural protein nanoplatforms from laboratory to clinical trials remains an enormous challenge. More efforts should be made to improve the NP reproducibility, colloidal stability in biological environments, and drug-loading efficiency. Furthermore, live and spleen accumulation is a big barrier in clinical translation of all types of NPs. Accordingly, more understanding of bio-nano interactions is required, while more data of pharmacokinetics should be collected and analyzed. Long-term toxicity of nanoformulation is not well evaluated. In addition to mouse model, more animal models, e.g., beagle dogs, monkeys and gorillas, should serve for assessing the long-term toxicity and therapeutic effect. Engineering complex particles with multiple functions are currently achieved by chemists. Accurate diagnose of early stage of tumor and precision drug delivery to tumor site should be more considerable in the future developing of protein-based cancer theranostics.

AUTHOR CONTRIBUTIONS
YG, HZ, and GS: designed this work of review; DM, MZ, and LW: performed the literature search of the databases; YG: wrote the manuscript; HZ and GS: revised the manuscript. All authors approved the paper for publication.