Sequential Vaccination With Heterologous Influenza HA Elicit Broad Cross-reactive Neutrlaization Antibodies

Objective:Influenza is a respiratory infectious disease that spreads between birds,mammals and human,with highly mortality among children,elderly and other high-risk populations.Influenza virus vaccines are the main precautionary measure.Although a variety of influenza vaccines have been developed and commencialized,influenza vaccine candidate strains need to be constantly updated due to the continuous drifting and mutating of influenza virus.Furthermore,the current influenza vaccines fail to mount broadly neutralizing antibodies.This study is intended to develop vaccines that can elicit broad cross-reactive neutralization antibodies against a variety of influenza viruses.In the first section,consensus H1,H3,and H5 antigen were deduced by the alignment of all published HA amino acid sequences from 2006 to 2009,and designated CON H1,CON H3 and CON H5 respectively.DNA vaccines were constructed based on these consensus sequences,designated as pVKD1.0-CON H1,pVKD1.0-CON H3 and pVKD1.0-CON H5 respectively,and were verified by double enzyme digestion,sequencing and in vitro protein expression.Animal experiments confirmed that the vaccines were highly immunogenic.In the second section,we mimicthe nature state of multiple infections by different influenza virusesand design the animal experimental scheme of sequential immunization with these three vaccines.To assess the immunogenicity of the sequential immunization of influenza vaccines,we compared immune responses following sequential immunizations with the CON H1,CON H3,and CON H5 vaccines,as well as after vaccination using a mixture of the CON H1,CON H3,and CON H5 vaccines.We next quantified binding antibody responses,hemagglutination inhibition(HAI)antibody responses,broadly neutralizing antibody(nAb)response,and then isolated broadly neutralizing monoclonal antibody(mAb)from sequentially vaccinated mice.Methods:Part one.Construction and assessment of DNA vaccines based on the consensus sequences of H1,H3 and H5 immunogensCON H1,CON H3 and CON H5 were reduced to the nucleotide sequence with mammalian codon usage frequency by the back translation softwareand added with restriction enzyme cutting sites,termination codones and corresponding restriction sites.These artificial synthetized consensus nucleotide sequences were chemically synthesized by Shanghai Generay Biotech Co,Ltd and then inserted into the DNA vaccine vector pVKD1.0,resulting three DNA vaccine vectors,designated pVKD1.0-CON H1,pVKD1.0-CON H3 and pVKD1.0-CON H5 respectively.The constructed vaccines were performed double enzyme digestion for verification and then were transfected into 293 T cells with Lipofectamine 2000 TM,after 24 hours culture,Western Bloting was performed to detect protein expression.BALB/c mice were intramuscularly administered the DNA vaccines or empty vectors 100μg at 0,2 and 4 weeks respectively.Two weeks after the final vaccine inoculation,the mice were sacrificed,and their splenocytes were harvested.ELISPOT were performed to determine cell immunogenicity based on peptide HA533(IYSTVASSL)and HA438(SYNAELLVAL).ELISA were performed to determine humoral immunogenicity based on HA1(BR07 H1),HA1(BR07 H3)and HA1(HK97H5)antigen.Vaccination groups were compared with control group and statistically processed by T test.Part two.Assay of the immunogenicity,hemagglutination inhibition and neutralizationof sequential vaccination regimenThe experimental animals were divided into three groups: sequential immunization group,mixed group and mock control group.The mice of sequential immunization group were injectedeach vaccine 100μg at anterior tibial muscle at 0,4 and 8 week respectively.The mixed immune group were injectedthe mixture of the three vaccines99μg(each 33μg)at 0,4 and 8 week respectively.The mock control group was vaccinated empty vector 100μg per injection.In tenth weeks the mice were extracted with inner canthus blood before sacrificed by cervical dislocation and the spleen cells were collected.Cellular immunogenicity was detected by ELISPOT based on IFN-γ and peptideHA533(IYSTVASSL).Humoral immunogenicity was detecting by ELISA based on HA protein and inactivate viruses derived from H1(BR07 H1、TJ09 H1 and SH09 H1),H3(BR07 H3)and H5(HK97 H5 and NV05 H5)subtype.Hemagglutination inhibition(HAI)antibody reaction was deteced by 4 inactivate viruse strains,including two H1(TJ09 H1、SH09 H1),one H3(SH09 H3)and one H5(NV05 H5)strains.Neutralization assay was conducted based on pseudoviruses,including H1(TE09H1,SI06 H1,SI86 H1 and BR07 H1),H3(MO99 H3,FJ02 H3,WI05 H3 andBR07 H3),and H5(VN04 H5)strains.To investigate whether the neutralization activity of sera from immunized mice was mediated by immunoglobulin,especially immunoglobulin G(IgG),several serum samples from four vaccinated mice were selected and quantified nAb titer before and after the depletion of IgG.We next vaccinated several BALB/c female mice by following the sequential regimen,the splenocytes were then collected from immunized mice two weeks after the last vaccination and were fused with SP2/0-Ag14 myeloma cell lines to generate clonal hybridomas cell lines.About 3200 hybridomas cell lines were screened with neutralization assays against all above pseudoviral strains respectively.To confirm the protective effect of sequential immunization,we implemented a challenge experiment with BAL B/c mice.Specific pathogen free(SPF)female 6-8week old BALB/c mice(Shanghai Slaccas,China)were administered intramuscularly(i.m.)above DNA vaccines.Two weeks after the last immunization mice were intranasally challenged with PR8 virus(A/Puerto Rico/8/1934(H1N1)),at a dose equivalent to 100 TCID50.Survival and body weight loss were monitored for 14 days.Results:Part one.Construction of DNA vaccine pVKD1.0-CON H1、pVKD1.0-CON H3 and pVKD1.0-CON H5.The constructed plasmids were underwent double enzyme digestion,electrophoresis results showed that the enzyme digestion products of three plamids were in the vicinity of 5KB,corresponding with pVCD1.0 vector fragment size;The products without enzyme digestion were in the vicinity of 7KB,corresponding with inserting gene sequence size in 2KB.The recombinant DNA vaccines were transfected in 293 T cell,transfection products were conducted by SDS-PAGE electrophoresis and Western Blot detection,the results showed that a obvious strip was at above 70 KD on pre-stainedproteinmarker,comparable to the expected 90 kD.Celluarimmunogenicity detecting: afterstimulating mouse spleen cells with peptide HA533(IYSTVASSL),the difference of IFN-γ dots between pVKD-CON H1 group and control group was significant(P < 0.01);the difference of IFN-γdots between pVKD-CON H5 group and empty vector group was significant(P<0.0001).After stimulating mouse spleen cells with peptide HA438(SYNAELLVAL),the difference ofIFN-γdots between pVKD-CON H3 group and empty vector group was significant(P< 0.05).Humoral immunogenicity detecting: the serum binding antibody titer of pVKD1.0-CON H1 group and pVKD1.0-CON H5 group both significantly higher than control group(P<0.01)against HA1(BR07 H1).The serum binding antibody titer of pVKD1.0-CON H3 group was significantlyhigher than control group(P<0.0001)against HA1(BR07 H3).The serum binding antibody titer of pVKD1.0-CON H5 group was significantly higher than control group(P<0.0001)against HA1(HK97 H5).In this experiment,pVKD1.0-CON H5 can activate BR07 H1 in a certain level,the most possible reason is,H5 and H1 subtype are belong to the first group of influenza A virus,so compare to H3 subtype they are more homologous.Part two.Assay of the immunogenicity,hemagglutination inhibition and neutralization of sequential vaccination regimenWe first tested the cellular immune response of immunized mice by IFN-γ based ELISPOT with a dominant epitope peptide HA533(IYSTVASSL).Compared to control mice(Mock group,11±5 Spot Forming Cells(SFCs)/106 splenocytes),all vaccine immunized groups were induced significantly more cellular responses(p<0.0001 for Seq.group vs Mock group;p<0.0001 for mixed group vs Mock group)(Fig.2A),sequential immunization group(Seq.group,948±54 SFCs//106 splenocytes)had the similar level of cellular response to the mixed immunization group(Mix.group,985±192 SFCs/106 splenocytes).Sequential immunization group had the similar level of cellular response to the mixed immunization group.We tested humoral immunogenicity based on ELISA.For H1 subtype,both regimens induced high levels of binding antibody titers against SH09 H1 strain(both p<0.05 compared to Mock group),but weak response against BR07 H1(both p<0.05 compared to Mock group)and against TJ09 H1(both p>0.05 compared to Mock mice).These data could be explained since CON H1 amino acid sequence was more homologous with SH09 H1 than BR07 H1 or TJ09 H1.Furthermore,three times ofimmunization of CON H1 in mixing group raised significant more binding antibodies against SH09 H1 than that in sequential regimen(p<0.0001).In contrast,for heterologous BR07 H1,sequential regimen was superior to mixing regimen(p<0.05).For H3 subtype,we assayed serum binding antibody titer against H3N2,the results showed that both regimens induced moderate and comparable antibody responses.The binding antibody responses against H5 subtype were similar to SH09 H1,significant antibodies were induced in both immunization groups(p<0.05,compared to Mock mice),and mixing regimen elicited significantly higher binding antibodies than sequential regimen.We further tested the hemagglutination inhibition(HAI)antibody responses in immunized mice with four inactivated strains,including two H1 strains,one H3 strain and one H5 strain.Significant HAI antibody responses were induced for all four strains except for TJ09 H1 and the response pattern was consistent with bindingantibody response.For SH09 H1,HAI response elicited by mixing regimen(69.6(47.4-102.3))was higher than sequential one(20.0(10.9-36.8))(p<0.01),coincided with the levels of binding antibody response.For other two strains,two regimens raised a similar level of HAI antibody responses.We evaluated serum neutralizing activity of all immunized mice against a panel of influenza virus subtypes and strains,including H1,H3,and H5 strains with pseudovirus-based neutralization assay.Among all four strains of H1 subtype,owing to the high homogeneity between CON H1 and the HA of TE09 H1,both sequential and mixing immunization groups elicited significant more nAb response against TE09 H1 for seq.group and for mixing group,compared to Mock mice(p<0.0001 for seq group vs mock and p<0.0001 for mixing group vs mock group),with slightly lower nAb titers in seq.group than that in mixing group.However,for other three heterologous H1 strains,mixing regimen failed to induce any nAb responses whereas sequential immunization indeed induced moderate but broadly serum nAb responsesfor SI86 H1,SI06 H1,and for BR07 H1 respectively compared to Mock group(p<0.05).We then determined nAb responses against four H3 subtype viruses,and observed that both vaccination regimens could induce significant nAb responses for all four H3 strains.For BR07 H3,the most homologous strain to vaccinated HA CON H3,both sequential and mixing immunization could elicit robust nAb responses.For all other three heterologous H3 strains,sequential group consistently elicited higher nAbresponses than mixing group and reached significance for titers against MO99 H3(the most heterologous strain to HA CON H3)(p<0.05)and against WI05 H3(p<0.001).Both regimens induced high levels of nAb titers against H5 subtype.Similar to binding antibodies,mixing group elicited higher nAb than sequential group against either VN04 H5(p<0.01)or NV05 H5(p<0.001).Furthermore,we determined nAb responses against other heterosubtypes(including H2,H7,H8,H9,H11,H12,H14 and H15 pseudovirus)with pooled serum,but no neutralizing activity was identified in sera from neither vaccination regimen groups.To investigate whether the neutralization activity of sera from immunized mice was mediated by immunoglobulin,especially immunoglobulin G(IgG),several serum samples from four vaccinated mice were selected and quantified nAb titer before and after the depletion of IgG with Protein G Sepharose Beads.As expected,the depletion of IgG resulted in the disappearance of the neutralization activities against all tested strains,including TE09 H1,BR07 H3,and VN04 H5.Therefore,the results confirmed that the neutralization activities were mainly mediated by sera IgG.We next vaccinated several BALB/c female mice by following the sequential regimen,the splenocytes were then collected from immunized mice two weeks after the last vaccination and were fused with SP2/0-Ag14 myeloma cell lines to generate clonal hybridomas cell lines.About 3200 hybridomas cell lines were screened with neutralization assays against all above pseudoviral strains respectively.Most hybridomas supernatant could only neutralize only strains which had a HA highly homologous to CON H1 or CON H3 amino acid sequence,including TE09 H1,SC18H1,BR07 H3,WI05 H3,or FJ02 H3.However,after two round confirmations,we finally isolated four potent broadly neutralizing mAbs,including 5A3-F1,5A3-F2,7B6-G11,6A7-F1.5A3-F1 and 5A3-F2 were isolated by screening with SC18 H1 pseudovirus and had broadly neutralizing activities against all tested H1 subtype virus and H5 subtype virus.Impressively,both the calculated IC50 of 5A3-F1 and 5A3-F2 against H1 subtype approached microgram level,and was even as low as 96.9 ng/mL against SC18 H1 and 11.1 ng/mL against TE09 H1.Surprisingly,5A3-F1 and 5A3-F2 both could also neutralize H5 subtype virus,and the neutralizing activities against these H5 viruses also approached microgram level.The calculated IC50 of 5A3-F1 were 3.0μg/mL,2.0 μg/mL,1.1 μg/mL,6.7 μg/mL for VN04 H5,NV05 H5,IN05 H5,XJ06 H5respectively;and the calculated IC50 of 5A3-F2 were 2.7 μg/mL,1.7 μg/mL,1.2 μg/mL,7.1 μg/mL for VN04 H5,NV05 H5,IN05 H5),XJ06 H5 respectively.In addition,5A3-F1(IC50: 1.0μg/mL)and 5A3-F2(IC50: 0.5μg/mL)could neutralize H3 subtype strains(FJ02 H3),and 5A3-F2 could even have weak neutralizing activity against H8virus(IC50: 2.0μg/mL).For other subtypes,both 5A3-F1 and 5A3-F2 had no neutralizing activity.The results of challenge experiment show that,In the Mock group,all mice`experienced largely body loss and on day 11,only one mice(1/6)was still alive.Mice immunized with both sequential and mixing vaccines were well protected,and most immunized mice had a remarkable recovery from weight loss.These results suggest good protection of sequential immunization against PR8 virus.However,because of the cross-reactivity between pandemic 2009 H1N1 strain and PR8 strain and the highly homogeneity of CON H1 with TE09 H1,Mix.group which had more CON H1 injections showed a full protection from infection and a better over sequential vaccination against the lethal infection of PR8 virus.The result was consistwith previously better binding antibody or nAb response against homologous virus such as TH09 H1,VN04 H5 and NV05 H5.Conclusions:1.DNA vaccines constructed with consensus H1,H3 and H5 are capable of inducing vigorous humoral and cellular immunity.2.Sequential immunization with Con H1,Con H3 and Con H5 is able to elicit broadly cross-reactive neutralization antibodies in comparison with traditional mixed immunization.3.Broad cross-reactive neutralizing antibody clones could be raised by sequential immunization and isolated for usage in treatment in future.