Difluoromethylornithine (DFMO), an Inhibitor of Polyamine Biosynthesis, and Antioxidant N-Acetylcysteine Potentiate Immune Response in Mice to the Recombinant Hepatitis C Virus NS5B Protein.

Hepatitis C virus (HCV) is one of the main triggers of chronic liver disease. Despite tremendous progress in the HCV field, there is still no vaccine against this virus. Potential vaccines can be based on its recombinant proteins. To increase the humoral and, especially, cellular immune response to them, more effective adjuvants are needed. Here, we evaluated a panel of compounds as potential adjuvants using the HCV NS5B protein as an immunogen. These compounds included inhibitors of polyamine biosynthesis and urea cycle, the mTOR pathway, antioxidants, and cellular receptors. A pronounced stimulation of cell proliferation and interferon-γ (IFN-γ) secretion in response to concanavalin A was shown for antioxidant N-acetylcysteine (NAC), polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO), and TLR9 agonist CpG ODN 1826 (CpG). Their usage during the immunization of mice with the recombinant NS5B protein significantly increased antibody titers, enhanced lymphocyte proliferation and IFN-γ production. NAC and CpG decreased relative Treg numbers; CpG increased the number of myeloid-derived suppressor cells (MDSCs), whereas neither NAC nor DFMO affected MDSC counts. NAC and DFMO suppressed NO and interleukin 10 (IL-10) production by splenocytes, while DFMO increased the levels of IL-12. This is the first evidence of immunomodulatory activity of NAC and DFMO during prophylactic immunization against infectious diseases.

Structure-based drug designing and immunoinformatics approach for SARS-CoV-2.

The prevalence of respiratory illness caused by the novel SARS-CoV-2 virus associated with multiple organ failures is spreading rapidly because of its contagious human-to-human transmission and inadequate globalhealth care systems. Pharmaceutical repurposing, an effective drug development technique using existing drugs, could shorten development time and reduce costs compared to those of de novo drug discovery. We carried out virtual screening of antiviral compounds targeting the spike glycoprotein (S), main protease (Mpro), and the SARS-CoV-2 receptor binding domain (RBD)-angiotensin-converting enzyme 2 (ACE2) complex of SARS-CoV-2. PC786, an antiviral polymerase inhibitor, showed enhanced binding affinity to all the targets. Furthermore, the postfusion conformation of the trimeric S protein RBD with ACE2 revealed conformational changes associated with PC786 drug binding. Exploiting immunoinformatics to identify T cell and B cell epitopes could guide future experimental studies with a higher probability of discovering appropriate vaccine candidates with fewer experiments and higher reliability.

Japanese encephalitis virus: A multi-epitope loaded peptide vaccine formulation using reverse vaccinology approach.

Japanese encephalitis (JE) is a serious leading health complication emerging expansively that has severely affected the survival rate of human beings. This fatal disease is caused by JE Virus (JEV). The current study was carried out for designing a multi-epitope loaded peptide vaccine to prevent JEV. Based on reverse vaccinology and in silico approaches, octapeptide B-cell and hexapeptide T-cell epitopes belonging to five proteins, viz. E, prM, NS1, NS3 and NS5 of JEV were determined. Hydrophilicity, antigenicity, immunogenicity and aliphatic amino acids of the epitopes were estimated. Further, the epitopes were analyzed for different physicochemical parameters, e.g. total net charges, amino acid composition and Boman index. Out of all the epitopes, a total of four T-cell epitopes namely KRADSS, KRSRRS, SKRSRR and KECPDE and one B-cell epitope i.e. PKPCSKGD were found to have potential for raising immunity in human against the pathogen. Taking into account the outcome of this study, the pharmaceutical industries could initiate efforts to combine the identified epitopes together with adjuvant or carrier protein to develop a multi-epitope-loaded peptide vaccine against JEV. The peptide vaccine, being cost effective, could be administered as a prophylactic measure and in JEV infected individuals to combat the spread of this virus in human population. However, prior to administration into human beings, the vaccine must pass through several clinical trials.

Attenuation and efficacy of live-attenuated Rift Valley fever virus vaccine candidates in non-human primates.

Rift Valley fever virus (RVFV) is an important mosquito-borne veterinary and human pathogen that has caused large outbreaks of severe disease throughout Africa and the Arabian Peninsula. Currently, no licensed vaccine or therapeutics exists to treat this potentially deadly disease. The explosive nature of RVFV outbreaks and the severe consequences of its accidental or intentional introduction into RVFV-free areas provide the impetus for the development of novel vaccine candidates for use in both livestock and humans. Rationally designed vaccine candidates using reverse genetics have been used to develop deletion mutants of two known RVFV virulence factors, the NSs and NSm genes. These recombinant viruses were demonstrated to be protective and immunogenic in rats, mice, and sheep, without producing clinical illness in these animals. Here, we expand upon those findings and evaluate the single deletion mutant (ΔNSs rRVFV) and double deletion mutant (ΔNSs-ΔNSm rRVFV) vaccine candidates in the common marmoset (Callithrix jacchus), a non-human primate (NHP) model resembling severe human RVF disease. We demonstrate that both the ΔNSs and ΔNSs-ΔNSm rRVFV vaccine candidates were found to be safe and immunogenic in the current study. The vaccinated animals received a single dose of vaccine that led to the development of a robust antibody response. No vaccine-induced adverse reactions, signs of clinical illness or infectious virus were detected in the vaccinated marmosets. All vaccinated animals that were subsequently challenged with RVFV were protected against viremia and liver disease. In summary, our results provide the basis for further development of the ΔNSs and ΔNSs-ΔNSm rRVFV as safe and effective human RVFV vaccines for this significant public health threat.

Antibody Epitopes Identified in Critical Regions of Dengue Virus Nonstructural 1 Protein in Mouse Vaccination and Natural Human Infections.

Dengue is a global public health problem and is caused by four dengue virus (DENV) serotypes (DENV1-4). A major challenge in dengue vaccine development is that cross-reactive anti-DENV Abs can be protective or potentially increase disease via Ab-dependent enhancement. DENV nonstructural protein 1 (NS1) has long been considered a vaccine candidate as it avoids Ab-dependent enhancement. In this study, we evaluated survival to challenge in a lethal DENV vascular leak model in mice immunized with NS1 combined with aluminum and magnesium hydroxide, monophosphoryl lipid A + AddaVax, or Sigma adjuvant system+CpG DNA, compared with mice infected with a sublethal dose of DENV2 and mice immunized with OVA (negative control). We characterized Ab responses to DENV1, 2, and 3 NS1 using an Ag microarray tiled with 20-mer peptides overlapping by 15 aa and identified five regions of DENV NS1 with significant levels of Ab reactivity in the NS1 + monophosphoryl lipid A + AddaVax group. Additionally, we profiled the Ab responses to NS1 of humans naturally infected with DENV2 or DENV3 in serum samples from Nicaragua collected at acute, convalescent, and 12-mo timepoints. One region in the wing domain of NS1 was immunodominant in both mouse vaccination and human infection studies, and two regions were identified only in NS1-immunized mice; thus, vaccination can generate Abs to regions that are not targeted in natural infection and could provide additional protection against lethal DENV infection. Overall, we identified a small number of immunodominant regions, which were in functionally important locations on the DENV NS1 protein and are potential correlates of protection.

Generation of monoclonal antibodies against native viral proteins using antigen-expressing mammalian cells for mouse immunization.

BACKGROUND: Due to their rising incidence and progressive geographical spread, infections with mosquito-borne viruses, such as dengue (DENV), chikungunya and zika virus, have developed into major public health challenges. Since all of these viruses may cause similar symptoms and can occur in concurrent epidemics, tools for their differential diagnosis and epidemiological monitoring are of urgent need. RESULTS: Here we report the application of a novel strategy to rapidly generate monoclonal antibodies (mAbs) against native viral antigens, exemplified for the DENV nonstructural glycoprotein 1 (NS1). The described system is based on the immunization of mice with transfected mammalian cells expressing the target antigens in multiple displays on their cell surface and thereby presenting them efficiently to the host immune system in their native conformation. By applying this cell-based approach to the DENV NS1 protein of serotypes 1 (D1NS1) and 4 (D4NS1), we were able to rapidly generate panels of DENV NS1 serotype cross-reactive, as well as D1NS1- and D4NS1 serotype-specific mAbs. Our data show that the generated mAbs were capable of recognizing the endogenous NS1 protein in DENV-containing biological samples. CONCLUSION: The use of this novel immunization strategy, allows for a fast and efficient generation of hybridoma cell lines, producing mAbs against native viral antigens. Envisaged applications of the mAbs include the development of test platforms enabling a differentiation of the DENV serotypes and high resolution immunotyping for epidemiological studies.

Tandem truncated rotavirus VP8* subunit protein with T cell epitope as non-replicating parenteral vaccine is highly immunogenic.

The two currently available live oral rotavirus vaccines, Rotarix(®) and RotaTeq(®), are highly efficacious in the developed countries. However, the efficacy of such vaccines in resource deprived countries in Africa and Southeast Asia is low. We reported previously that a bacterially-expressed rotavirus P2-P[8] ΔVP8* subunit vaccine candidate administered intramuscularly elicited high-titers of neutralizing antibodies in guinea pigs and mice and significantly shortened the duration of diarrhea in neonatal gnotobiotic pigs upon oral challenge with virulent human rotavirus Wa strain. To further improve its vaccine potential and provide wider coverage against rotavirus strains of global and regional epidemiologic importance, we constructed 2 tandem recombinant VP8* proteins, P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8] ΔVP8*-P[6] ΔVP8* based on Escherichia coli expression system. The two resulting recombinant tandem proteins were highly soluble and P2-P[8] ΔVP8*-P[8] ΔVP8* was generated with high yield. Moreover, guinea pigs immunized intramuscularly by 3 doses of the P2-P[8] ΔVP8*-P[8] ΔVP8* or P2-P[8] ΔVP8*-P[6] ΔVP8* vaccine with aluminum phosphate adjuvant developed high titers of homotypic and heterotypic neutralizing antibodies against human rotaviruses bearing G1-G4, G8, G9 and G12 with P[8], P[4] or P[6] combination. The results suggest that these 2 subunit vaccines in monovalent or bivalent formulation can provide antigenic coverage to almost all the rotavirus G (VP7) types and major P (VP4) types of global as well as regional epidemiologic importance.

Inclusion of a universal tetanus toxoid CD4(+) T cell epitope P2 significantly enhanced the immunogenicity of recombinant rotavirus ΔVP8* subunit parenteral vaccines.

Currently available live oral rotavirus vaccines, Rotarix(®) and RotaTeq(®), are highly efficacious in developed countries. However, the immunogenicity and efficacy of such vaccines in some developing countries are low. We reported previously that bacterially-expressed rotavirus ΔVP8* subunit vaccine candidates with P[8], P[4] or P[6] specificity elicited high-titer virus neutralizing antibodies in animals immunized intramuscularly. Of note was the finding that antibodies induced with the P[8]ΔVP8* vaccine neutralized both homotypic P[8] and heterotypic P[4] rotavirus strains to high titer. To further improve its vaccine potential, a tetanus toxoid universal CD4(+) T cell epitope P2 was introduced into P[8] or P[6]ΔVP8* construct. The resulting recombinant fusion proteins expressed in Escherichia coli were of high solubility and were produced with high yield. Two doses (10 or 20 µg/dose) of the P2-P[8]ΔVP8* vaccine or P2-P[6]ΔVP8* vaccine with aluminum phosphate adjuvant elicited significantly higher geometric mean homologous neutralizing antibody titers than the vaccines without P2 in intramuscularly immunized guinea pigs. Interestingly, high levels of neutralizing antibody responses induced in guinea pigs with 3 doses of the P2-P[8]ΔVP8* vaccine persisted for at least 6 months. Furthermore, in the gnotobiotic piglet challenge study, three intramuscular doses (50 µg/dose) of the P2-P[8]ΔVP8* vaccine with aluminum phosphate adjuvant significantly delayed the onset of diarrhea and significantly reduced the duration of diarrhea and the cumulative diarrhea score after oral challenge with virulent human rotavirus Wa (G1P[8]) strain. The P2-P[8]ΔVP8* vaccine induced serum virus neutralizing antibody and VP4-specific IgG antibody production prechallenge, and primed the pigs for higher antibody and intestinal and systemic virus-specific IFN-γ producing CD4(+) T cell responses postchallenge. These two subunit vaccines could be used at a minimum singly or preferably in bivalent formulation to provide antigenic coverage of most of the G types of global importance.

Surface modified microprojection arrays for the selective extraction of the dengue virus NS1 protein as a marker for disease.

While advances in assay chemistry and detection continue to improve molecular diagnostics technology, blood samples are still collected using the 150-year-old needle/syringe method. Surface modified microprojection arrays have been developed as a novel platform for in vivo, needle-free biomarker capture. These devices are gold coated silicon arrays with >20,000 projections per cm(2), which can be applied to the skin for tunable penetration into the epidermis or dermis. The microprojection array conceptually offers several advantages over the current methods including: minimally invasive sample collection, no need for sample processing and concentration of specific markers at the device surface for sensitive detection. In this study, Microprojection arrays were coated with antibodies to capture an early marker of dengue virus infection, NS1, from the skin of live mice. We also developed a complementary "total IgG" assay which could be used as a positive control for adequate penetration of the projections. Surface modifications designed for selective extraction were tested against standard microtiter plate ELISA. We also investigated the use of Protein G-mediated antibody immobilization in order to orient capture antibodies. While we found that capture efficiency could be improved, the direct EDC-based antibody immobilization resulted in a significantly higher surface density leading to a higher degree of NS1 capture. Using mice intravenously injected with recombinant dengue virus type 2 NS1 as a pseudomodel for dengue infection, NS1 was successfully extracted using microprojection arrays sampling from skin fluid, with a detection limit of 8 µg/mL.

Neutralizing antibodies against rotavirus produced in transgenically labelled purple tomatoes.

Edible fruits are inexpensive biofactories for human health-promoting molecules that can be ingested as crude extracts or partially purified formulations. We show here the production of a model human antibody for passive protection against the enteric pathogen rotavirus in transgenically labelled tomato fruits. Transgenic tomato plants expressing a recombinant human immunoglobulin A (hIgA_2A1) selected against the VP8* peptide of rotavirus SA11 strain were obtained. The amount of hIgA_2A1 protein reached 3.6 ± 0.8% of the total soluble protein in the fruit of the transformed plants. Minimally processed fruit-derived products suitable for oral intake showed anti-VP8* binding activity and strongly inhibited virus infection in an in vitro virus neutralization assay. In order to make tomatoes expressing hIgA_2A1 easily distinguishable from wild-type tomatoes, lines expressing hIgA_2A1 transgenes were sexually crossed with a transgenic tomato line expressing the genes encoding Antirrhinum majus Rosea1 and Delila transcription factors, which confer purple colour to the fruit. Consequently, transgenically labelled purple tomato fruits expressing hIgA_2A1 have been developed. The resulting purple-coloured extracts from these fruits contain high levels of recombinant anti-rotavirus neutralizing human IgA in combination with increased amounts of health-promoting anthocyanins.

[Preclinical studies of live intranasal H5N1 influenza vaccine with the deleted HS1 gene].

The paper gives the results of evaluating the efficiency of deINS1 pandemic H5N1 vaccine candidate VN1203delNS1 which was constructed by reverse genetics on the basis of influenza virus strain A/Vietnam/1203/04. The safety, immunogenicity and cross-protection of the vaccine strain against different H5N1 virus clades were demonstrated in mouse and macaque models. The results showed the possibility of designing a new-generation replication-deficient intranasal influenza vaccine, by applying an approach to deleting the NS1 pathogenicity factor, an antagonist of the interferon system.

Inhibition of protease-inhibitor-resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies.

Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons resistant to small-molecule inhibitors of the NS3/4A protease, and replicons derived from different HCV genotypes. The combination of intrabodies and interferon-α had an additive inhibitory effect on RNA replication in the replicon model. Intrabody expression also inhibited production of infectious HCV in a cell culture system. The NS3 protease activity was inhibited by the intrabodies in NS3-expressing cells. In contrast, cell-free synthesis of HCV RNA by preformed replicase complexes was not inhibited by intrabodies, suggesting that the major mode of inhibition of viral replication is inhibition of NS3/4A protease activity and subsequent suppression of viral polyprotein processing.

Rational vaccine strategies against AIDS: background and rationale.

New vaccine candidates exploiting the rational combination of regulatory and structural HIV gene products are being developed within the program of the AIDS Vaccine Integrated Project (AVIP) and will be tested in comparative preclinical and clinical trials with the ultimate goal of selecting proper candidates for advanced clinical testing in developing countries.

HCV NS3 serine protease-neutralizing single-chain antibodies isolated by a novel genetic screen.

Hepatitis C virus (HCV) infection is a major world-wide health problem causing chronic hepatitis, liver cirrhosis and primary liver cancer. The high frequency of treatment failure points to the need for more specific, less toxic and more active antiviral therapies for HCV. The HCV NS3 is currently regarded as a prime target for anti-viral drugs, thus specific inhibitors of its activity are of utmost importance. Here, we report the development of a novel bacterial genetic screen for inhibitors of NS3 catalysis and its application for the isolation of single-chain antibody-inhibitors. Our screen is based on the concerted co-expression of a reporter gene, of recombinant NS3 protease and of fusion-stabilized single-chain antibodies (scFvs) in Escherichia coli. The reporter system had been constructed by inserting a short peptide corresponding to the NS5A/B cleavage site of NS3 into a permissive site of the enzyme beta-galactosidase. The resulting engineered lacZ gene, coding for an NS3-cleavable beta-galactosidase, is carried on a low copy plasmid that also carried the NS3 protease-coding sequence. The resultant beta-galactosidase enzyme is active, conferring a Lac+ phenotype (blue colonies on indicator 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-gal) plates), while induction of NS3 expression results in loss of beta-galactosidase activity (transparent colonies on X-gal plates). The identification of inhibitors, as shown here by isolating NS3-inhibiting single-chain antibodies, expressed from a compatible high copy number plasmid, is based on the appearance of blue colonies (NS3 inhibited) on the background of colorless colonies (NS3 active). Our source of inhibitory scFvs was an scFv library that we prepared from spleens of NS3-immunized mice and subjected to limited affinity selection. Once isolated, the inhibitors were validated as genuine and specific NS3 binders by an enzyme-linked immunosorbent assay and as bone fide NS3 serine protease inhibitors by an in vitro catalysis assay. We further show that upon expression as cytoplasmic intracellular antibodies (intrabodies) in NS3-expressing mammalian cells, three of the scFvs inhibit NS3-mediated cell proliferation. Although applied here for the isolation of antibody-based inhibitors, our genetic screen should be applicable for the identification of candidate inhibitors from other sources.

A plant-based multicomponent vaccine protects mice from enteric diseases.

Cholera toxin (CT) B and A2 subunit complementary DNAs (cDNAs) were fused to a rotavirus enterotoxin and enterotoxigenic Escherichia coli fimbrial antigen genes and transferred into potato. Immunoblot and enzyme-linked immunosorbent assay (ELISA) results indicated that the fusion antigens were synthesized in transformed tuber tissues and assembled into cholera holotoxin-like structures that retained enterocyte-binding affinity. Orally immunized mice generated detectable levels of serum and intestinal antibodies against the pathogen antigens. Elevated levels of interleukin 2 (IL2) and interferon gamma (INFgamma) detected in immunogen-challenged spleen cells from the immunized mice indicated the presence of a strong Th1 immune response to the three plant-synthesized antigens. This result was supported by flow cytometry analysis of immunized mouse spleen cells that showed a significant increase in CD4+ lymphocyte numbers. Diarrhea symptoms were reduced in severity and duration in passively immunized mouse neonates following rotavirus challenge. The results suggest that food plants can function as vaccines for simultaneous protection against infectious virus and bacterial diseases.

Antibodies to parvovirus B19 non-structural protein are associated with chronic but not acute arthritis following B19 infection.

OBJECTIVE: To determine the incidence and significance of antibodies to the parvovirus B19 non-structural (NS1) protein in B19-infected persons during acute infection and convalescence. METHODS: The B19 NS1 protein was expressed in SF9 cells using the baculovirus expression system and was used to prepare immunofluorescence slides. These were used in a fluorescent antibody test to determine anti-B19 NS1 IgG in a well-characterized cohort of 53 persons at the time of acute B19 infection and again after a follow-up period of 26-85 months. Results were examined for statistical significance by the use of Fisher's exact test. RESULTS: NS1 antibodies were detected in five of 32 persons with acute B19 infection (four with arthritis) and 10 of 53 persons with past B19 infection (six with chronic arthritis and two with chronic arthritis and chronic fatigue syndrome). Regarding the correlation of NS1 antibodies and arthritis, at the time of acute infection four of 24 persons with arthritis had NS1 antibodies detected compared with one of eight persons with any other symptoms (P: = 1). During convalescence, eight of 20 persons with chronic arthritis had NS1 antibodies compared with two of 33 with symptoms of any other category (all except one were asymptomatic) (P: = 0.007). All 10 patients with NS1 antibodies during convalescence had arthritis during acute infection, which persisted in eight persons until the time of follow-up. CONCLUSION: Antibodies to parvovirus B19 NS1 protein are associated with chronic but not with acute arthritis after B19 infection.

[A comparison of the immune response induced by DNA or by an inactivated vaccine against tick-borne encephalitis]. / Sravnenie immunnogo otveta, indutsirovannogo DNK ili inaktivirovannoi vaktsinoi protiv kleshchevogo éntsefalita.

BALB/c mice were immunized with recombinant plasmid DNA pSVK3-ENS1 and pcDNAI-NS3 containing, respectively, genes E-NS1 and NS3 of tick-borne encephalitis (TBE) virus. Antibodies to TBE virus proteins were detected in the blood sera of the immunized animals by the method of the enzyme immunoassay. Though the titers of virus-specific antibodies in the sera of mice immunized with protein vaccines exceeded those registered after immunization with DNA vaccines, essential protective immunity was observed after the use of both vaccines.

Immune responses to hepatitis C virus structural and nonstructural proteins induced by plasmid DNA immunizations.

DNA-based immunizations have been used to elicit cellular immunity to hepatitis C virus (HCV) proteins in mice. Mice were immunized by intramuscular or intradermal injections of plasmid DNA derived from a near-full-length HCV genotype 1b genomic clone (pRC/B2) or individual genomic clones. These immunizations induced cytotoxic T lymphocytes (CTLs), as revealed in standard chromium-release assays that used syngeneic peptide-pulsed or transfected target cells. These assays identified four CTL epitopes within the capsid, E1, and E2 regions of the polyprotein sequence of HCV genotype 1a that were cross-reactive with HCV genotype 1b. Additionally, CTLs derived from mice immunized with either NS3 or NS5 specifically lysed target cells sensitized to either the genotype 1a or 1b gene products. Nucleic acid immunizations also generated humoral immunity to HCV proteins, as detected by anti-HCV reactivity to NS3 and capsid in ELISAs and immunoblot assays.

Recombinant, chimaeric live, attenuated vaccine (ChimeriVax) incorporating the envelope genes of Japanese encephalitis (SA14-14-2) virus and the capsid and nonstructural genes of yellow fever (17D) virus is safe, immunogenic and protective in non-human primates.

Yellow fever 17D virus, a safe and effective live, attenuated vaccine, was used as a vector for genes encoding the protective antigenic determinants of a heterologous member of the genus Flavivirus, Japanese encephalitis (JE) virus, the leading cause of acute viral central nervous system infection and death throughout Asia. The viral envelope (prM and E) genes of a full-length cDNA clone of YF 17D virus were replaced with the corresponding genes of JE SA14-14-2, a strain licensed as a live, attenuated vaccine in China. Full-length RNA transcripts of the YF/JE chimaera were used to transfect Vero cells. The progeny virus (named 'ChimeriVax-JE'), was used to define safety after intracerebral (i.c.) inoculation of rhesus monkeys. Monkeys (N = 3) inoculated with a high dose (6.6 log10 pfu) developed a brief viremia, showed no signs of illness, developed high titers of anti-JE neutralizing antibody, and had minimal brain and spinal cord lesion scores according to criteria specified in the WHO monkey neurovirulence test. A control group of 3 monkeys that received a lower dose (4.2 log10 pfu) of commercial YF 17D vaccine had slightly higher lesion scores. To develop a lethal monkey model of JE for vaccine protection tests, we inoculated groups of monkeys i.c. or intranasally (i.n.) with a JE virus strain found to be highly neurovirulent and neuroinvasive for mice. Monkeys inoculated i.c., but not i.n., developed severe encephalitis after an incubation period of 8-13 days. The ChimeriVax-JE virus was passed in a cell line acceptable for human use (diploid fetal rhesus lung) and 4.3 or 5.3 log10 pfu were inoculated into groups of 3 monkeys by the subcutaneous route. All 6 animals developed brief viremias (peak titer < 2.0 log10 pfu/ml) and subsequently had anti-JE but no yellow fever neutralizing antibodies. On day 64, the monkeys were challenged i.c. with 5.5 log10 pfu of virulent JE virus. The immunized animals had no detectable viremia post-challenge, whereas 4 unimmunized controls became viremic. Only 1 of 6 (17%) vaccinated monkeys but 4 of 4 (100%) unvaccinated controls developed encephalitis. Histopathological examination 30 days after challenge confirmed that the protected, immunized animals had no or minimal evidence of encephalitis. These data demonstrated the ability of the ChimeriVax-JE to induce a rapid humoral immune response and to protect against a very severe, direct intracerebral virus challenge. Target areas of neuronal damage and inflammation in monkeys infected IC with wild-type JE, the chimaeric virus and YF 17D were similar, indicating that the histopathological scoring system used for the WHO yellow fever monkey neurovirulence test will be applicable to control testing of chimaeric seed viruses and vaccines.