Characterization Of Non-neutralizing Antibody Response Against Enteroviruses

Enterovirus(EV) is an important genus in Picornaviridae family. This genus is phylogenically divided into 12 species, containing over 300 different serotypes infecting humans and aninals frequently. After eradicating of the infection of polioviruses, hand, foot and mouth diseases(HFMD) mainly caused by enterovirus 71(EV71), coxsackievirus A16(CA16) and other enteroviruses such as CA6, CA10, CA5, CA10 and Coxsackievirus B3(CB3) has become the most serious diseases and has led to millions of attacks and several outbreaks across the world and become more predominant in Asia-Pacific Region, especially in Mainland C hina. HFMD usually attacks children under the age of five causing various clincal symptoms and even death, putting a serious threat on public health. There were still no effective anti- virus therapies and avaliable vaccines for HFMD.Host humoral immunity plays a key role in the defense of viral infection, however, the epidemic law of EVs and how humans defend and and clear EV infection remains unknown so far, bringing many difficulties in effective prevention and treatment of disease. Studies concerning the host humoral immune responses against infection of EVs are primarily based on the neutralizing antibody assay. Not only neutralizing antibody, but also non-neutralizing antibody could be produced after EV infection. Was the neutralizing antibody response prevalent in the infection of EVs? Whether the non- neutralizing antibody has the same dynamic change as neutralizing antibody with the increase of age, how the neutralizng antibody was produced and whether it has an impact on neutralizing antibody remain unclear. These questions were studied in our experiments to further understand the characterization and significance of non-neutralizing antibody response and provide new sight for fully understand the interaction with host and EVs. The main contents were as follows. 1. Characterization of non-neutralizing antibody response against EV71 capsid proteins in Chinese individuals by NEIBM-ELISAIn this study, the DNA sequence of EV71 VP0, VP3, VP1 and truncated VP1 antigens(VP11-60, VP145-58, VP141-297, VP161-297 and VP1134-297) were obtained by PCR with the plasmid EV71 VP0-p MD-18 T, VP3-p MD-18 T and VP1-p MD-18 T as templates. The recombinant protein were harvested through prokaryotic expression and then purified with the Ni-NTA affinity chromatography. A NEIBM(novel evolved immunoglobulin-binding molecule)-based ELISA(N EIBM-ELISA) method was established to analyze anti-EV71 non-neutralizing antibody response in humans. 33 serum specimens of severe HFMD cases and 194 clinical serum samples of non-HFMD children cases were also collected as the age- match controls from the Wuxi people’s hospital, Jiangsu, China. 200 serum specimens were collected as normal adult controls from healthy blood donors at C hanghai Hospital, Shanghai, China. The anti-EV71 VP1, VP0 and VP3 non- neutralizing antibody response in serum samples from severs HFMD cases and normal adults were detected using NEIBM-ELISA method. To estimate the contribution of full length VP1 and various truncated VP1 proteins to the anti-EV71 VP1 reaction, ELISA and competitive ELISA were performed using full- length and truncated VP1 antigens in serum samples with high anti-VP1 antibody response from three groups.The high seroprevalence of anti-EV71 capsid proteins(VP1, VP0 and VP3) was found in both normal adults and severe HFMD cases, the seroprevalence and reactivity of anti-EV71 VP1 was also significantly stronger than that of anti-EV71 VP0 and VP3 in normal adults and severe HFMD cases, respectively. VP145-58, VP141-297 and VP11-60 contained the common enterovirus cross-reactive sequence(C ECRS) showed obvious reactivity with the VP1 strongly reactive samples, while VP161-297 and VP1134-297 without CECRS did not show obvious reactivity. Consistently, VP141-297, VP11-60 and VP145-58 also exhibited significantly higher level of inhibition than VP161-297 lacking of CEC RS. The inhibition patterns to anti- EV71 VP1 antibody response by various VP1 antigens also exhibited the different characteristics between three groups.These results indicated the high prevalence of antibody responses against all three capsid proteins in humans. and anti- EV71 capsid antibody response is predominately stimulated against the VP1 protein. Anti- EV71 VP1 antibody response was found to predominantly target to epitopes based on the CEC RS. We here proposed that VP1 epitopes based on CECRS could be classified into three kinds of antigens: core antigen(VP145-58), N antigen(VP11-60) and C antigen(VP141-297). The full VP1 antigen consists of these three antigens and other epitopes without C ECRS. The antibody response characterized in the present study was different from that of neutralizing antibody, and we hypothesized the antibody response against VP1 epitopes based on CEC RS characterized in this study represents the major host antibody response to EV71 infection and could highly cross react with the EV71 related viruses, while the neutralizing antibody response represents the minor antibody response which shows high specificity and little cross reactivity. This finding should have some importance for vaccine development. 2. Characterization of non-neutralizing antibody response against VP1 proteins of various enteroviruses in Shanghai childrenThe gene sequence of VP1 of EV71 and CA16 from EV-A, CB3 from EV-B, PV1(human poliovirus1) from EV-C and RV13(human rhinovirus A13) from RV-A were acquired through sequential OE-PCR and reverse-transcription PCR method. The recombinant plasmids of p ET-21b-EV71 VP1, p ET-21b-CA16 VP1, p ET-21b-CB3 VP1, p ET-21b-PV1VP1 and p ET-21b-RV13 VP1 were constructed and recombinant proteins were induced and purified. 364 serum samples from Shanghai outpatient children of various ages(<28days, 1-6months, 6-12 m, 12-24 m, 24-36 m, 36-48 m, 48-60 m and 60-72m)without symptoms of HFMD were collected. NEIBM- ELISA and competitive inhibition ELISA were performed to detect the anti-VP1 non- neutralizing antibody response of various enteroviruses. Statistical significance and correlation analysis of the reactivity and inhibition of the antibody response were performed.The non-neutralizing antibody reactivity against VP1 of various EVs showed a similar age-related dynamic change among infants and children. The non-neutralizing antibodies agaisnt various EVs were high in the neonates(<28d), then became lowest between 1 to 12 months of age, then reached a peak level between 48 to 60 months of age, and decreased remarkably during 60-72 months(except RV13 VP1). This dynamic can be divided into three stages(< 28 d, 1-12 m and 12-72m) to compare the reactivity and the inhibition level of non-neutralizing antibody response against VP1 of five enteroviruses. The level of anti-VP1 of RV-A at 1-28 days of age was significantly higher than that of anti- VP1 of other detected viruses, demonstrated the highest level; the levels of anti-VP1 of C B3 and PV1 were significantly higher than that of anti-VP1 of EV71 and CA16, showed the second high level; and the levels of anti-VP1 of EV71 and CA16 showed the low level. The anti-VP1 levels of all detected viruses at 12-72 months of age were as same high as these at 1-28 days of age. The correlation coefficients of antibody response against five VP1 proteins in two ages(12-72 m and <28d) showed three levels: the highest level in EV-A(between enteroviruses), the second highest level between EV-A, B and C, and the lowest level between RV-A and EV-A, B, C. The correlation coefficients of inhibition to anti-VP1 reaction of five EVs had no correlation with sequence homology, varied between 1-28 days and 12-72 months of age and declined in 12-72 m comparing to <28d.These results indicated the non- neutralizing antibody response in neonates(<28days of age) represented the maternal-derived antibody responses, the antibody responses at 12-72 months of age reflected the primary antibody responses, the antibody responses at 1 to 12 months of age represented the converted antibody responses from maternally-derived antibody responses to primary antibody responses. The anti- VP1 levels of all detected EVs at 1-28 days of age were as same high as these at 12-72 months of age which were significantly higher than these at 1-12 months of age. Moreover, the order of anti-VP1 levels of all detected viruses from high to low(RV-A > C B3 > PV1 > CA16 > EV71) at 1-28 days was same as that at 12-72 months, indicating the non-neutralizing antibody response against EVs(RV13) transmitted through respiratory tract was higher than that of EVs transmitted through intestinal tract; and in EVs transmitted through intestinal tract, the antibody response of EV-B(CB3) was higher than than that of EV-C(PV1) and EV-A, and the antibody response against EV71 was higher than that againtst CA16 in EV-A. The correlation of anti-VP1 response in 12-72 months and 1-28 days of age reflected the sequence homology of various VP1 proteins. The correlation of inhibition to anti-VP1 reaction of five EVs was attributed to sequence homology and the antibody response. The distinction of the inhibition level and correlation coefficients reflected different non-neutralizing antibody response between 1-28 days and 12-72 months of age. 3. Identification of diffe rent non-neutrlizing antibody response against the CA16 VP1 capsid protein: specific or non-specific antibody responseThe DNA sequence of full- length and truncated CA16 VP1 antigens(VP11-297,VP11-60, VP145-58, VP141-297 and VP161-297) were obtained by PCR with the plasmid CA16 VP1-p MD-18 T as a template. The recombinant prokaryotic expression vector were constructed. The recombinant protein were harvested through prokaryotic expression and then purified. ELISA and competitive inhibition ELISA analysis using five C A16 VP1 antigens were conducted to characterize the non-neutralizing antibody response against CA16 VP1 in serum samples with high anti-CA16 VP1 antibody response from the Shanghai blood donors and Shanxi elderly individuals. Statistical significance and correlation analysis of the reactivity and inhibition of the antibody response were performed. The neutralizing titers of the serum samples from different inhibition types of the two groups were also detected.In this study, the reactivity to VP141-297 was significantly higher compared to VP161-297, and all of the CECRS-based antigens(VP11-297, VP141-297, VP11-60 and even VP145-58) showed significantly higher inhibition potency than VP161-297 in samples from the Shanghai blood donors. In the Shanxi individuals, although the reactivity level against VP141-297 was not significantly different compared to the reactivity level against VP161-297, the inhibition level of VP141-297 was significantly higher compared to VP161-297. The correlation between reaction of various CA16 VP1 antigens and the inhibition to anti-VP1 reaction in Shanghai blood donors was obviously higher than that in Shanxi individuals. We characterized anti-CA16 VP1 reaction based on the inhibition degrees by VP11-60 and VP141-297 to define 11 types of inhibition profile to anti-CA16 VP1 reaction. There were 6 types of inhibition profile in the Shanghai blood donors including the defined specific inhibition type which showed as the predominant type and accounted for 71.4% of the samples. Differently, so many as 9 types of inhibition profile were found in the Shanxi individuals, and the biggest type of inhibition only accounted for 28.3% of the samples, while the specific inhibition type accounted for only 6.5%. Consistently, the neutralizing antibody titre in the samples defined as the specific inhibition type in the Shanghai blood donors was significantly higher compared to that in the samples of other inhibition types, indicating specific CA16 infection.This study demonstrated that the non-neutralizing antibody response against CA16 VP1 was also able to mainly respond to epitopes based on the common enterovirus cross-reactive sequence in the Shanghai blood donors. Different anti-CA16 VP1 antibody response and inhibition pattern were found in two groups. The correlation between reaction of various CA16 VP1 antigens(and especially inhibition to anti-CA16 VP1 antibody response by various antigens) in Shanghai blood donors was obviously higher than that in Shanxi individuals, exhibited a potentially consiste nt, specific anti-CA16 antibody response in Shanghai blood donors. A predominant inhibition type was found in Shanghai blood donors, and the neutralizing antibody response was obviously higher than other inhibition types, indicating the specific antibody response caused by infection of CA16. The anti-CA16 antibody response in Shanxi elderly individuals exhibited a non-specific antibody response caused by infection of other EVs. The correlation coefficients of reaction against VP141-297 and VP11-297 could be an effective index for the evaluation of the degree of consistency of anti-CA16 VP1 reaction, and thus the high correlation coefficient could indicate the epidemic degree of a specific C A16 infection in individuals. These results might provide a potential basis for the identification of an effective approach for the seroepidemiological surveillance of C A16 infection based on ELISA. 4. Study on induction of non-neutralizing antibody response in mice and the impact on neutralizing antibody responseRecombinant VP1 proteins of p ET21b-EV71, p ET21b-CA16, p ET21b-CA5, p ET21b-CA6 、 p ET21b-CA10, p ET21b-CB3, p ET21b-PV1 and p ET21b-RV13 were expressed and purified. Mice were immunized with various recombinant VP1(except EV71 or CA16 VP1) proteins to construct a cross-infection model to simulate multiple infection of EVs in humans were defined as EV71 or C A16 cross-reaction group, and mice merely immunized with EV71 or CA16 VP1 protein were defined as EV71 or CA16 group. Negative control groups were immunized with BSA or nothing. Mice were bled before primary immunization and 14 days after each immunization. Full- length and truncated antigens of EV71 and CA16 VP1 were expressed and purified as previously. NEIBM-ELISA and competitive inhibition ELISA were performed to detect the antibody response against EV71 /CA16 VP1 antigens during the process of cross- inoculation. On the basis of the mouse model, the same doses of inactivated EV71 /CA16 vaccine were inoculated in mice of cross-reaction and control groups two times at 2-week intervals, and individual serum samples were collected 2 weeks after primary immunization and lasted for 2-9 weeks at 1-week interval. The neutralizing antibody against EV71 or CA16 were detected using micro- neutralizing assay to compare the titers and duration of the neutralizing antibody response between cross-reaction and control groups.During the construction of mouse model through innoculation with VP1 from different EVs, with the increase of immunization, anti-EV71 VP11-60 gradually approached and exceeded anti- EV71 VP161-297 and anti- EV71 VP141-297 gradually approached anti-EV71 VP11-297. The non-neutralizing antibody response against EV71 was shifted to targeted to epitopes based on CECRS. However, in EV71 group, anti- EV71 antibody response was in proportion to the length of EV71 VP1 antigens. Competitive inhibition ELISA also showed EV71 antigens containing CEC RS could well inhibit anti- EV71 VP1 non-neutralizing antibody response in EV71 cross-reaction group. Four types o f inhibition to anti-EV71 and anti-CA16 VP1 antibody response by various VP1 proteins in EV71 cross-reaction group and EV71 group exhibited different characteristics, and were found in Shanghai neonates with 1-28 days of age. These results indicated inoculation with VP1 proteins of various EVs could successfully induce the non-neutralizing antibody response similar in humans. The anti-VP1 antibody response caused by infection of one kind of EV could cross react with VP1 of other EVs, and infection with multiple EVs could result in the shift of non- neutralizing antibody response to epitopes based on C ECRS.On the basis of the mouse model, inactivated vaccines were inoculated and the neutralizing antibody titers were detected. After inoculating with EV71 inactivated vaccine, the titers and duration of neutralizing antibody against EV71 in cross reaction and control groups showed no significant difference. The anti-CA16 neutralizing antibody in CA16 cross-reaction group, CA16 group and negative control group showed no significant difference after primary inoculation of C A16 vaccine and the titers all increased after the second inoculation. Moreover, the anti-CA16 neutralizing antibody in negative group tended to exceed that in CA16 cross-reaction group and CA16 group, and the anti-CA16 neutralizing antibody in C A16 cross-reaction group tended to be lower than C A16 group. These results indicated the non-neutralizing antibody has little impact on inducing neutralizing antibody response against EV71, but might affect the induction of anti-CA16 neutralizing antibody response. Conclusions:In our study, anti- EV71 VP1 antibody response was firstly found to predominantly target to epitopes based on the CEC RS and the concept of non- neutralizing antibody response was proposed. The non- neutralizing antibody response against various EVs was found to be prevalent in normal adults, elderly people, patients infected with HFMD and children with different ages. The age-related dynamic change of non-neutralizng antibody response against EVs was firstly and systematically observed in infants and children from 1 day to 6 years of age and the concept of materal-derived Ig G and primary antibody respons was raised. The non-neutralizing antibody response against EV71/CA16 VP1 could be successfully induced in mouse model, and the potential formation mechanism of non-neutralizing antibody response were found that infections of multiple EVs could lead to the shift of non-neutralizing antibody to epitopes based on CEC RS. The clues were observed in mouse model that presence of non-neutralizing antibody response might have different impact on the production of neutralizing antibody response against EV71 or CA16. Our study on non-neutralizng antibody response could be helpful for the better understanding of anti-EVs humoral immunity and provide useful information for seroepidemiological surveillance of EV infection.