Newcastle disease virus-based MERS-CoV candidate vaccine elicits high-level and lasting neutralizing antibodies in Bactrian camels

Middle East respiratory syndrome coronavirus （MERS-CoV）, a member of the Coronavifidae family, is the causative pathogen for MERS that is characterized by high fever, pneumonia, acute respiratory distress syndrome （ARDS）, as well as extrapul- monary manifestations. Currently, there are no approved treatment regimens or vaccines for MERS. Here~ we generated recombinant nonvirulent Newcastle disease virus （NDV） LaSota strain expressing MERS-CoV S protein （designated as rLa- MERS-S）, and evaluated its immunogenicity in mice and Bactrian camels. The results revealed that rLa-MERS-S showed similar growth properties to those of LaSota in embryonated chicken eggs, while animal immunization studies showed that rLa-MERS-S induced MERS-CoV neutralizing antibodies in mice and camels. Our findings suggest that recombinant rLa- MERS-S may be a potential MERS-CoV veterinary vaccine candidate for camels and other animals affected by MERS.

Transforming growth factor -β neutralizing antibodies inhibit subretinal fibrosis in a mouse model

AIM:To determine the involvement of the transforming growth factor(TGF)-β with the development of experimental subretinal fibrosis in a mouse model.· METHODS:Subretinal fibrosis was induced by subretinal injection of macrophage-rich peritoneal exudate cells(PECs) and the local expression of TGF-β isoforms was assessed by quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR) and enzyme-linked immunosorbent assay(ELISA) at various time points.In addition,we investigated the effect of TFG-β-neutralizing antibodies(TGF-β NAb) on subretinal fibrosis development.· RESULTS:TGF-β1 and TGF-β2 mRNA level was significantly elevated at day 2 after subretinal fibrosis induction and increased further to 5 and 6.5-fold respectively at day 5,reaching the peak.TGF-β3 mRNA was not detected in the present study.The result of ELSIA showed that active TGF-β1 and TGF-β2 levels were upregulated to 10-fold approximately,while total TGF-β1 and TGF-β2 levels were even upregulated more than 10-fold and more than 20-fold respectively in subretinal fibrosis mice in comparison with na?觙ve mice at day 5.TGF-β NAb resulted in a reduced subretinal fibrosis areas by 65% compared to animals from control group at day 7.· CONCLUSION:Our results indicate that TGF-β signaling may contribute to the pathogenesis of subretinal fibrogenesis and TGF-β inhibition may provide an effective,novel treatment of advanced and late-stage neovascular age-related macular degeneration.·

Detection of HPV Types and Neutralizing Antibodies in Women with Genital Warts in Tianjin City,China

The serum samples and corresponding cervical swabs were collected from 50 women with genital warts from Tianjin city, China. The neutralizing antibodies against HPV-16, -18, -58, -45, -6 and -11 in serum samples were tested by using pseudovirus-based neutralization assays and HPV DNAs in cervical swabs were also tested by using a typing kit that can detect 21 types of HPV. The results revealed that 36% （18/50） of sera were positive for type-specific neutralizing antibodies with a titer range of 160-2560, of which 22%（11/50）, 12%（6/50）, 10%（5/50）, 4%（2/50）, 4%（2/50） and 2%（1/50） were against HPVs -6, -16, -18, -58, -45 and -1 l, respectively. Additionally, 60% （30/50） of samples were HPV DNA-positive, in which the most common types detected were HPV-68（18%）, HPV-16（14%）, HPV-58（12%）, HPV-33（8%） and HPV-6, HPV-11, HPV-18 and HPV-52 （6% each）. The concordance between HPV DNA and corresponding neutralizing antibodies was 56% （28/50） with a significant difference （P〈0.05）. The full-length sequences of five HPV types （HPV -42, -52, -53, -58 and -68） were determined and exhibited 98%-100% identities with their reported genomes. The present data may have utility for investigating the natural history of HPV infection and promote the development of HPV vaccines.

Neutralizing antibodies in hepatitis C virus infection

Hepatitis C virus (HCV) is a major cause of hepatitis world-wide. The majority of infected individuals develop chronic hepatitis which can then progress to liver cirrhosis and hepatocellular carcinoma. Spontaneous viral clearance occurs in about 20%-30% of acutely infected individuals and results in resolution of infection without sequaelae. Both viral and host factors appear to play an important role for resolution of acute infection. A large body of evidence suggests that a strong, multispecific and long-lasting cellular immune response appears to be important for control of viral infection in acute hepatitis C. Due too the lack of convenient neutralization assays, the impact of neutralizing responses for control of viral infection had been less defined. In recent years, the development of robust tissue culture model systems for HCV entry and infection has finally allowed study of antibody-mediated neutralization and to gain further insights into viral targets of host neutralizing responses. In addition, detailed analysis of antibody-mediated neutralization in individual patients as well as cohorts with well defined viral isolates has enabled the study of neutralizing responses in the course of HCV infection and characterization of the impact of neutralizing antibodiesfor control of viral infection. This review will summarize recent progress in the understanding of the molecular mechanisms of antibody-mediated neutralization and its impact for HCV pathogenesis.

Safe and Objective Assay of Enterovirus 71 Neutralizing Antibodies via Pseudovirus

Current serum neutralization assays based on the inhibition of the cytopathic effect（Nt-CPE） need to ma nipulate live viruses, which are time-consuming, labor-intensive, and have the potential exposure to infectious agents, so a safe and objective assay via pseudovirus for the fast and efficient detection of enterovirus 71（EV71） neutralizing antibodies was developed. First, we generated EV71 pseudovirus containing firefly luciferase gene in place of the capsid gene P1 in EV71 genome. Vero cells infected with 200 CCID50（50% cell culture infective dose） of EV71 pseudovirus for 24 h were found to have the best performance. Seval sera were measured by EV71 pseudoparticle neutralization assay（Nt-PPN） and the conventional serological method Nt-CPE. Neutralizing antibody titers measured by Nt-PPN and those obtained by Nt-CPE demonstrate a high correlation between the two methods. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of EV71 neutralizing anti bodies. This method can be used for detecting neutralizing antibodies of other picornaviruses, such as hepatitis A vi rus（HAV） and coxsackievirus 16（CVA16）, and make it possible to determine whether there is cross-reactivity be tween EV71 and CVA16.

An unusual COVID-19 case with over four months of viral shedding in the presence of low neutralizing antibodies: a case report

The ongoing coronavirus disease 2019(COVID-19)pandemic is a global public health crisis,causing social and economic disasters in many countries.In China,two-consecutive negative results of nucleic acid tests for SARS-CoV-2 from the respiratory samples are required to end the quarantine of COVID-19 patients.However,clinicians face a dilemma in case of patients with long-term viral shedding.This report described an unusual COVID-19 case who had persistent viral RNA positivity for more than 4 months after initial illness in the presence of low neutralizing antibodies,but without prolonged clinical symptoms.Multiple anti-viral drug treatments had no impact and there was no evidence of re-infection.When the patient was self-quarantined at home,no infection occurred to the three family members living with her for 15 to 19 days.Sputum viral culture in BSL-3 laboratory on the 102nd day after symptom onset was negative.From the 129th day on,8 continuous nucleic acid tests of sputum samples showed negative results.The patient was discharged on 137th days since symptom onset.In conclusion,viral RNA shedding in the sputum of the COVID-19 patient may last over 4 months.As no evidence shows the existence of infectious virus,two-consecutive negative nucleic acid tests may not be the prerequisite for ending quarantine of COVID-19 patients with prolonged viral shedding.

“Unconventional”Neutralizing Activity of Antibodies Against HIV

Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by inununization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity, heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using ＂conventional＂ neutralization assay which uses phytohemagglutinin （PHA）-stimulated human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4^＋T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera, although they do not have neutralizing activity when tested by the ＂conventional＂ neutralization assay, do exhibit potent and broad neutralizing activity in ＂unconventional＂ ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages and inunature dendritic cells （iDCs）, or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.

Epitope Analysis of Anti-SARS-CoV-2 Neutralizing Antibodies

Coronavirus disease 2019 is threatening thousands of millions of people around the world.In the absence of specific and highly effective medicines,the treatment of infected persons is still very challenging.As therapeutics,neutralizing antibodies(NAbs)have great potential.Many NAbs have been reported,and most target various regions on the receptor-binding domain of the spike(S)protein,or the N-terminal domain.Several NAbs and NAb cocktails have been authorized for emergency use,and more arc in clinical trials or are under development.In this review,considering the angle of binding epitopes on the S protein,we summarize the functions and the underlying mechanisms of a set of well-recognized NAbs and provide guidance for vaccine design and the combinatorial use of these antibodies.In addition,we review the NAbs and NAb cocktails that have been approved for emergency use and discuss the effectiveness of these NAbs for combating severe acute respiratory syndrome coronavirus 2 mutants.

Cross-neutralizing Anti-hemagglutinin Antibodies Isolated from Patients Infected with Avian Influenza A(H5N1) Virus

Objective To recover broad-neutralizing monoclonal antibodies(Bn Abs)from avian influenza A(H5N1)virus infection cases and investigate their genetic and functional features.Methods We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients.The genetic basis,biological functions,and epitopes of the obtained Bn Abs were assessed and modeled.Results Two Bn Abs,2-12 D5,and 3-37 G7.1,were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset.Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity(ADCC)activity.Albeit derived from distinct Ab lineages,i.e.,V^H1-69-D2-15-JH^4(2-12D5)and V^H1-2-D3-9-JH^5(3-32 G7.1),the Bn Abs were directed toward CR6261-like epitopes in the HA stem,and HA2 I45 in the hydrophobic pocket was the critical residue for their binding.Signature motifs for binding with the HA stem,namely,IFY in VH1-69-encoded Abs and LXYFXW in D3-9-encoded Abs,were also observed in 2-12D5 and 3-32 G7.1,respectively.Conclusions Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus.The HA stem epitopes targeted by the Bn Abs,and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.

Neutralizing possibilities of whole virion and mRNA vaccine triggered antibodies of Wuhan strain of SARS-CoV-2 with receptor binding domains of spike proteins of Delta and Omicron strains

Mutations in the receptor binding domain(RBD)of SARS-CoV-2 spike protein have given birth to the new variants of concern viz.Delta and Omicron strain.As we are using vaccines which are either mRNA of spike protein of Wuhan strain or its whole virion vaccine,these vaccines will trigger antibodies to be able to bind with the epitopes of spike protein of Wuhan strain.There appears a need to study whether vaccine generated circulating neutralizing antibodies in vaccinated people are able to neutralize the receptor binding domain of spike protein of currently circulating Delta and Omicron strains as well.We need to address this contention in two phases:first to undertake docking studies of vaccine generated antibodies with Wuhan,Delta and Omicron strains and second to conduct the wet lab studies and observe antigen-antibody bindings employing Immunofluorescence and Cryo-Electron Microscopic studies.The present paper reports results of docking studies using bioinformatic softwares to provide the baseline information for wet lab studies.

Challenges on Induction of Broadly Neutralizing Antibodies for Optimization of HIV Vaccines Development and Vectored Immunoprophylaxis

Despite extensive research efforts, a preventive human immunodeficiency virus (HIV) vaccine remains one of the major challenges in the field of AIDS research. Experimental strategies which have been proven successful for other viral vaccines are not enough to tackle HIV-1 and new approaches to design effective preventive AIDS vaccines are of utmost importance. Due to enormous diversity among global circulating HIV strains, an effective HIV vaccine must elicit broadly protective antibodies based responses;therefore discovering new broadly neutralizing antibodies (bNAbs) against HIV has become major focus in HIV vaccine research. However further understanding of the viral targets of such antibodies and mechanisms of action of bNAbs is required for advancement of HIV vaccine research. This technical note discusses our current knowledge on the bNAbs and immunoprophylaxis using viral vectors with their relevance in designing of new candidates to HIV-1 vaccines.

Broadly neutralizing antibodies target a hemagglutinin anchor epitope

Broadly neutralizing antibodies(bnAbs)targeting epitopes of the influenza virus hemagglutinin(HA)have the potential to provide near universal protection against influenza virus infection1.However,viral mutants that escape bnAbs have been reported2,3.The identification of bnAb classes that can neutralize viral escape mutants is critical for universal influenza virus vaccine design.Here,we report a distinct class of bnAbs targeting a discrete membrane-proximal anchor epitope of the HA stalk domain.Anchor epitope-targeting antibodies are broadly neutralizing across H1 viruses and can cross-react with pandemic-threat H2 and H5 viruses.Antibodies targeting this anchor epitope utilize a highly restricted repertoire,which encodes for two public binding motifs that make extensive contacts with conserved residues in the fusion peptide.Moreover,anchor epitope-targeting B cells are common in the human memory B cell(MBC)repertoire and were recalled in humans by an oil-in-water adjuvanted chimeric HA(cHA)vaccine4,5,a potential universal influenza virus vaccine.To maximize protection against seasonal and pandemic influenza viruses,vaccines should aim to boost this previously untapped source of bnAbs that are widespread in the human MBC pool.

Generation of antibodies against disintegrin and cysteine-rich domains by DNA immunization: An approach to neutralize snake venom-induced haemorrhage

Objective: To explore whether a DNA immunization approach targeting the major haemorrhage molecule of a prothrombin activator-like metalloproteinase from Echis ocellatus(E. ocellatus) venom could be conceived to inspire antibodies with more prominent specificity and equal adequacy to current conventional antivenoms systems.Methods: The isolated DNA Eo MP-6 was used as the template for PCR amplification using the Eo DC-2-specific forward and reverse primers. A PCR product of approximately700 bp was obtained and cloned into p Sec Tag-B expression vector where anti-Eo DC-2antibodies were generated and analysed for their efficacy to neutralise local haemorrhage in vitro and in vivo.Results: Our results suggest that the generated anti-Eo DC-2 showed a remarkable efficacy by(a) interfering with the interaction of the recombinant disintegrin "Eo DC-2" isolated from the E. ocellatus as well as other viper species to the a2b1-integrins on platelets;(b) complete inhibition of the catalytic site of the metalloproteinase molecules in vitro using an adaptation antibody zymography assay. Furthermore, it has a polyspecific potential and constitutively expressed significant inhibition by cross-reaction and neutralised venom-induced local haemorrhage exerted by different viper species in vivo. The potential characteristic of Eo DC-2 against one part(the non-catalytic domain) as opposed to the whole molecule to neutralise its haemorrhagic activity is of crucial importance as it represents a novel approach with greater immunological specificity and fewer hazards, if any, than conventional systems of antivenom production, by exposure large animals that usually being used for the current antivenom production to a less injurious than expression of the whole molecule containing the catalytic metalloprotease domain. Hence, we report for the first time that our preliminary results hold a promising future for antivenom development.Conclusions: Antibodies generated against the E. ocellatus venom prothrombin activatorlike metalloprotease and disintegrin-cysteine-rich domains modulated and inhibited the catalytic activity both in vitro and in vivo of venom metalloproteinase disintegrin cysteine rich molecules. Thus, generating of venom specific-toxin antibodies by DNA immunization offer a more rational treatment of snake envenoming than conventional antivenom.

In vivo neutralization assays by monoclonal antibodies against white spot syndrome virus in crayfish (Cambarus proclarkii)

The neutralizing activities of eight monoclonal antibodies （MAbs） against white spot syndrome virus （WSSV） （2D2, 2B2,1D2, 1D5, 1C2, 4A1, 6A4 and 6B4） were analyzed by in vivo experiments. Gills from WSSV-infected shrimp were homogenized and ten-fold serially diluted by PBS, and then incubated with MAbs （hybridoma culture supernatant）, respectively. The mixture of WSSV and MAbs were injected into crayfish （Procambarus clarkii）. After challenge, the death rates of crayfish were counted to determine the neutralizing activities of MAbs. At the same time, the mixture of myeloma culture supernatant and WSSV or PBS was served as positive or negative control, respectively. The results showed that, at each virus dilution, the mean time to death of the crayfish injected with MAb-treated virus was significantly longer than that in the positive control, though they all showed 100% mortality within 25 d, and meanwhile, few crayfish died in the negative control. Among the eight MAbs, 2D2, 2B2, 1D2 and 1D5, especially the former two, delayed the mortality significantly, and 1 C2, 4A1 and 6A4 delayed the mortality as well but not so efficiently, while MAb 6IM was efficient only when the virus concentration increased. The results indicated that the anti-WSSV MAbs can neutralize WSSV in different virus dilutions.

Effects of CXCR7-neutralizing antibody on neurogenesis in the hippocampal dentate gyrus and cognitive function in the chronic phase of cerebral ischemia

Stromal cell-derived factor-1 and its receptor CXCR4 are essential regulators of the neurogenesis that occurs in the adult hippocampal dentate gyrus.However,the effects of CXCR7,a new atypical receptor of stromal cell-derived factor-1,on hippocampal neurogenesis after a stroke remain largely unknown.Our study is the first to investigate the effect of a CXCR7-neutralizing antibody on neurogenesis in the dentate gyrus and the associated recovery of cognitive function of rats in the chronic stage of cerebral ischemia.The rats were randomly divided into sham,sham+anti-CXCR7,ischemia and ischemia+anti-CXCR7 groups.Endothelin-1 was injected in the ipsilateral motor cortex and striatum to induce focal cerebral ischemia.Sham group rats were injected with saline instead of endothelin-1 via intracranial injection.Both sham and ischemic rats were treated with intraventricular infusions of CXCR7-neutralizing antibodies for 6 days 1 week after surgery.Immunofluorescence staining with doublecortin,a marker for neuronal precursors,was performed to assess the neurogenesis in the dentate gyrus.We found that anti-CXCR7 antibody infusion enhanced the proliferation and dendritic development of doublecortin-labeled cells in the dentate gyrus in both ischemic and sham-operated rats.Spatial learning and memory functions were assessed by Morris water maze tests 30-32 days after ischemia.CXCR7-neutralizing antibody treatment significantly reduced the escape latency of the spatial navigation trial and increased the time spent in the target quadrant of spatial probe trial in animals that received ischemic insult,but not in sham operated rats.These results suggest that CXCR7-neutralizing antibody enhances the neurogenesis in the dentate gyrus and improves the cognitive function after cerebral ischemia in rats.All animal experimental protocols and procedures were approved by the Institutional Animal Care and Use Committee of China Medical University(CMU16089 R)on December 8,2016.

Staphylococcus aureusβ-hemolysin-neutralizing single-domain antibody isolated from phage display library of Indian desert camel

Objective:To isolate and characterize Staphylococcus aureus(S.aureus)β-hemolysinneutralizing dAbs from phage display library of Indian desert camel.Methods:Phage display library of 5×10 dAb clones of LPS-immunized Indian desert camel constructed in our laboratory was used for selection of S.aureus exotoxin-specific clones by panning technique.Enrichment of Ag-specific clones in successive rounds of panning was assessed by phage-ELISA and phage titration.Different dAb clones binding to S.aureus exotoxin Ags were expressed with C-terminal 6×His tag in E.coli and purified by Ni-chelate chromatography.The expression was verified by SDS-PAGE and western blotting.The purified clones were tested for inhibition of ’hot-cold’ hemolytic activity in vitro.Resistance to thermal inactivation of the dAb clones was studied by observing the effect of heat treatment from 50℃to 99℃for 30 min on the ’hot-cold’ hemolytic activity in vitro.Results:Several dAb clones binding to S.aureus exotoxins were isolated and enriched by three rounds of panning.The soluble dAb clones were approximately～16 kDa in size and reacted with 6×His tag specific murine monoclonal antibody in western blot.One of the Ni-chelate affinity purified dAb.6×His clones,inhibited S.aureusβ-hemolysin activity in vitro and resisted thermal inactivation upto 991.Conclusions:An S.aureusβ-hemolysinneutralizing dAb clone of possible therapeutic potential has been isolated.

Recent Developments in SARS-CoV-2 Neutralizing Antibody Detection Methods

The ongoing Coronavirus disease 19 pandemic has likely changed the world in ways not seen in the past.Neutralizing antibody(NAb)assays play an important role in the management of the severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)outbreak.Using these tools,we can assess the presence and duration of antibody-mediated protection in naturally infected individuals,screen convalescent plasma preparations for donation,test the efficacy of immunotherapy,and analyze NAb titers and persistence after vaccination to predict vaccine-induced protective effects.This review briefly summarizes the various methods used for the detection of SARS-CoV-2 NAbs and compares their advantages and disadvantages to facilitate their development and clinical application.

Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12

The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.

Rabies Virus Neutralizing Activity,Safety,and Immunogenicity of Recombinant Human Rabies Antibody Compared with Human Rabies Immunoglobulin in Healthy Adults

Objective Preliminary assessment of rabies virus neutralizing activity,safety and immunogenicity of a recombinant human rabies antibody(NM57)compared with human rabies immunoglobulin(HRIG)in Chinese healthy adults.Methods Subjects were randomly(1:1:1)allocated to Groups A(20 IU/kg NM57),B(40 IU/kg NM57),or C(20 IU/kg HRIG).One injection was given on the day of enrollment.Blood samples were collected on days-7 to 0(pre-injection),3,7,14,28,and 42.Adverse events(AEs)and serious AEs(SAEs)were recorded over a period of 42 days after injection.Results All 60 subjects developed detectable rabies virus neutralizing antibodies(RVNAs)(>0.05 IU/mL)on days 3,7,14,28,and 42.The RVNA levels peaked on day 3 in all three groups,with a geometric mean concentration(GMC)of 0.2139 IU/mL in Group A,0.3660 IU/mL in Group B,and0.1994 IU/mL in Group C.At each follow-up point,the GMC in Group B was significantly higher than that in Groups A and C.The areas under the antibody concentration curve over 0-14 days and 0-42 days in Group B were significantly larger than those in Groups A and C.Fifteen AEs were reported.Except for one grade 2 myalgia in Group C,the other 14 were all grade 1.No SAEs were observed.Conclusion The rabies virus neutralizing activity of 40 IU/kg NM57 was superior to that of 20 IU/kg NM57 and 20 IU/kg HRIG,and the rabies virus neutralizing activity of 20 IU/kg NM57 and 20 IU/kg HRIG were similar.Safety was comparable between NM57 and HRIG.

Human Enterovirus 71 DNA Vaccine Constructs Containing 5’UTR with Complete Internal Ribosome Entry Site Sequence Stimulated Improved Anti-Human Enterovirus 71 Neutralizing Immune Responses

Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for hand, foot and mouth disease (HFMD) have been developed. Here we examined the potential of improving the vaccines by inserting the EV71 5’ untranslated region (5’ UTR) containing the full length internal ribosome entry site (IRES) sequence to the EV71 VP1-based DNA vaccine constructs. Four vaccine constructs designated as 5’ UTR-VP1/EGFP, VP1/EGFP, 5’ UTR-VP1/pVAX and VP1/pVAX, were designed using the pEGFP-N1 and pVAX-1 expression vectors, respectively. Transfection of Vero cells with the vaccine constructs with the 5’-UTR (5’-UTR-VP1/EGFP and 5’ UTR-VP1/pVAX) resulted in higher percentages of cells expressing the recombinant protein in comparison to cells transfected with vectors without the 5’-UTR (67% and 57%, respectively). Higher IgG responses (29%) were obtained from mice immunized with the DNA vaccine construct with the full length 5’ UTR. The same group of mice when challenged with life EV71 produced significantly higher neutralizing antibody (NAb) titers (>5-fold). These results suggest that insertion of the EV71 5’ UTR sequence consisting of the full length IRES to the EV71 DNA vaccine constructs improved the efficacy of the constructs with enhanced elicitation of the neutralizing antibody responses.