An In-Depth Exploration of Mapping openEHR and PGHD: A Case Study on Fitbit-Generated Data.

In recent years, the adoption of wearable gadgets such as Fitbit has revolutionized the way individuals track and monitor their personal activity data. These devices provide valuable in-sights into an individual's physical activity levels, sleep patterns, and overall health metrics. Integrating this data into healthcare informatics systems can offer significant benefits in terms of personalized healthcare delivery and improved patient outcomes. This paper explores the synergistic integration of Fitbit-generated personal activity data using the openEHR Reference Model in healthcare informatics as a practical case study in patient-generated health data (PGHD) integration based on health informatics standards as a framework for the representation and exchange of Electronic Health Records (EHRs). The synergistic integration of Fitbit-generated personal activity data through openEHR and FHIR standards models also covers the way for advanced analytics and population health management. By linking and analyzing data from various sources, including sensors and wearable devices, healthcare organizations can identify trends, patterns, and insights that can guide population health strategies, preventive care initiatives, and personalized treatment plans, in addition to aiding physicians in follow-up care.

Validation of a Human Challenge Model Using an LT-Expressing Enterotoxigenic E. coli Strain (LSN03-016011) and Characterization of Potential Amelioration of Disease by an Investigational Oral Vaccine Candidate (VLA1701).

Controlled human infection models are important tools for the evaluation of vaccines against diseases where an appropriate correlate of protection has not been identified. Enterotoxigenic Escherichia coli (ETEC) strain LSN03-016011/A (LSN03) is an LT enterotoxin and CS17-expressing ETEC strain useful for evaluating vaccine candidates targeting LT-expressing strains. We sought to confirm the ability of the LSN03 strain to induce moderate-to-severe diarrhea in a healthy American adult population, as well as the impact of immunization with an investigational cholera/ETEC vaccine (VLA-1701) on disease outcomes. A randomized, double-blinded pilot study was conducted in which participants received two doses of VLA1701 or placebo orally, one week apart; eight days after the second vaccination, 30 participants (15 vaccinees and 15 placebo recipients) were challenged with approximately 5 × 109 colony-forming units of LSN03. The vaccine was well tolerated, with no significant adverse events. The vaccine also induced serum IgA and IgG responses to LT. After challenge, 11 of the placebo recipients (73.3%; 95%CI: 48.0-89.1) and 7 of the VLA1701 recipients (46.7%; 95%CI: 24.8-68.8) had moderate-to-severe diarrhea (p = 0.26), while 14 placebo recipients (93%) and 8 vaccine recipients (53.3%) experienced diarrhea of any severity, resulting in a protective efficacy of 42.9% (p = 0.035). In addition, the vaccine also appeared to provide protection against more severe diarrhea (p = 0.054). Vaccinees also tended to shed lower levels of the LSN03 challenge strain compared to placebo recipients (p = 0.056). In addition, the disease severity score was lower for the vaccinees than for the placebo recipients (p = 0.046). In summary, the LSN03 ETEC challenge strain induced moderate-to-severe diarrhea in 73.3% of placebo recipients. VLA1701 vaccination ameliorated disease severity, as observed by several parameters, including the percentage of participants experiencing diarrhea, as well as stool frequency and ETEC severity scores. These data highlight the potential value of LSN03 as a suitable ETEC challenge strain to evaluate LT-based vaccine targets (NCT03576183).

Safety and immunogenicity against ancestral, Delta and Omicron virus variants following a booster dose of an inactivated whole-virus COVID-19 vaccine (VLA2001): Interim analysis of an open-label extension of the randomized, controlled, phase 3 COV-COMPARE trial.

OBJECTIVES: Booster doses for COVID-19 vaccinations have been shown to amplify the waning immune response after primary vaccination and to enhance protection against emerging variants of concern (VoCs). Here, we aimed to assess the immunogenicity and safety of a booster dose of an inactivated whole-virus COVID-19 vaccine (VLA2001) after primary vaccination with 2 doses of either VLA2001 or ChAdOx1-S (Oxford-Astra Zeneca), including the cross-neutralization capacity against the Delta and Omicron VoCs. METHODS: This interim analysis of an open-label extension of a randomized, controlled phase 3 trial assessed a single booster dose of an inactivated whole-virus COVID-19 vaccine (VLA2001) in healthy or medically stable adults aged 18 years and above, recruited in 21 clinical sites in the UK, who had previously received two doses of either VLA2001 or ChAdOx1-S. Safety outcomes were frequency and severity of solicited injection site and systemic reactions within 7 days after booster vaccination as well as frequency and severity of any unsolicited adverse events (AE) after up to 6 months. Immunogenicity outcomes were the immune response to ancestral SARS-CoV-2 assessed 14 days post booster expressed as geometric mean titres (GMT), GMT fold ratios and seroconversion of specific neutralizing antibodies and S-protein binding IgG antibodies. Immunogenicity against the Delta and Omicron VoCs was assessed as a post-hoc outcome with a pseudovirus neutralization antibody assay. This study is registered with ClinicalTrials.gov, NCT04864561, and is ongoing. RESULTS: A booster dose of VLA2001 was administered to 958 participants, of whom 712 had been primed with VLA2001, and 246 with ChAdOx1-S. Within 7 days following these booster doses, 607 (63.4%) participants reported solicited injection site reactions, and 487 (50.8%) reported solicited systemic reactions. Up to 14 days post booster, 751 (78.4%) participants reported at least one adverse event. The tolerability profile of a booster dose of VLA2001 was similar in VLA2001-primed and ChAdOx1-S-primed participants. In VLA2001-primed participants, the GMT (95% CI) of neutralizing antibodies increased from 32.5 (22.8, 46.3) immediately before to 521.5 (413.0, 658.6) 2 weeks after administration of the booster dose, this corresponds to a geometric mean fold rise (GMFR) of 27.7 (20.0, 38.5). Compared to 2 weeks after the second priming dose, the GMFR was 3.6 (2.8, 4.7). In the ChAdOx1-S primed group, the GMT (95% CI) of neutralizing antibodies increased from 65.8 (43.9, 98.4) immediately before to 188.3 (140.3, 252.8) 2 weeks after administration of the booster dose, a geometric mean fold rise (GMFR) of 3.0 (2.2, 4.0). Compared to 2 weeks after the second priming dose, the GMFR was 1.6 (1.1, 2.2). For S-protein binding IgG antibodies, the pre- versus post-booster GMT fold ratio (95% CI) was 34.6 (25.0, 48.0) in the VLA2001-primed group and 4.0 (3.0, 5.2) in the ChAdOx1-S-primed group. Compared to 2 weeks after the second priming dose, the GMT fold rise of IgG antibodies was 3.8 (3.2, 4.6) in the VLA2001-primed group and 1.2 (0.9, 1.6) in the ChAdOx1-S-primed group. The GMT against Delta (B.1.617.2) and Omicron (BA.4/5) increased from 4.2 to 260, and from 2.7 to 56.7, respectively, when boosting subjects previously primed with VLA2001. Following the boost, 97% of subjects primed with VLA2001 had detectable Delta- and 94% Omicron-neutralizing antibodies. In subjects primed with ChAdOx1-S, the GMT against Delta and Omicron titres increased from 9.1 to 92.5, and from 3.6 to 12.3, respectively. After boosting, 99% of subjects primed with ChAdOx1-S had detectable Delta- and 70% Omicron-neutralizing antibodies. In both VLA2001 and ChAdOx1-S primed subjects, the additional VLA2001 dose boosted T cell responses against SARS-CoV-2 antigens to levels above those observed before the booster dose. CONCLUSION: A booster dose of VLA2001 was safe and well tolerated after primary immunization with VLA2001 and ChAdOx1-S. The tolerability of a booster dose of VLA2001 was similar to the favourable profile observed after the first and second priming doses. Both in a homologous and a heterologous setting, boosting resulted in higher neutralizing antibody titres than after primary immunization and significant increases in cross-neutralization titres against Delta and Omicron were observed after the booster dose. These data support the use of VLA2001 in booster programmes in ChadOx1-S primed groups.

Generation and genetic stability of tick-borne encephalitis virus mutants dependent on processing by the foot-and-mouth disease virus 3C protease.

Mature protein C of tick-borne encephalitis virus (TBEV) is cleaved from the polyprotein precursor by the viral NS2B/3 protease (NS2B/3(pro)). We showed previously that replacement of the NS2B/3(pro) cleavage site at the C terminus of protein C by the foot-and-mouth disease virus (FMDV) 2A StopGo sequence leads to the production of infectious virions. Here, we show that infectious virions can also be produced from a TBEV mutant bearing an inactivated 2A sequence through the expression of the FMDV 3C protease (3C(pro)) either in cis or in trans (from a TBEV replicon). Cleavage at the C terminus of protein C depended on the catalytic activity of 3C(pro) as well as on the presence of an optimized 3C(pro) cleavage site. Passage of the TBEV mutants bearing a 3C(pro) cleavage site either in the absence of 3C(pro) or in the presence of a catalytically inactive 3C(pro) led to the appearance of revertants in which protein C cleavage by NS2B/3(pro) had been regained. In three different revertants, a cleavage site for NS2B/3(pro), namely RR*C, was now present, leading to an elongated protein C. Furthermore, two revertants acquired additional mutations in the C terminus of protein C, eliminating two basic residues. Although these latter mutants showed wild-type levels of early RNA synthesis, their foci were smaller and an accumulation of protein C in the cytoplasm was observed. These findings suggest a role of the positive charge of the C terminus of protein C for budding of the nucleocapsid and further support the notion that TBEV protein C is a multifunctional protein.

The unique transmembrane hairpin of flavivirus fusion protein E is essential for membrane fusion.

The fusion of enveloped viruses with cellular membranes is mediated by proteins that are anchored in the lipid bilayer of the virus and capable of triggered conformational changes necessary for driving fusion. The flavivirus envelope protein E is the only known viral fusion protein with a double membrane anchor, consisting of two antiparallel transmembrane helices (TM1 and TM2). TM1 functions as a stop-transfer sequence and TM2 as an internal signal sequence for the first nonstructural protein during polyprotein processing. The possible role of this peculiar C-terminal helical hairpin in membrane fusion has not been investigated so far. We addressed this question by studying TM mutants of tick-borne encephalitis virus (TBEV) recombinant subviral particles (RSPs), an established model system for flavivirus membrane fusion. The engineered mutations included the deletion of TM2, the replacement of both TM domains (TMDs) by those of the related Japanese encephalitis virus (JEV), and the use of chimeric TBEV-JEV membrane anchors. Using these mutant RSPs, we provide evidence that TM2 is not just a remnant of polyprotein processing but, together with TM1, plays an active role in fusion. None of the TM mutations, including the deletion of TM2, affected early steps of the fusion process, but TM interactions apparently contribute to the stability of the postfusion E trimer and the completion of the merger of the membranes. Our data provide evidence for both intratrimer and intertrimer interactions mediated by the TMDs of E and thus extend the existing models of flavivirus membrane fusion.

Immunogenicity and safety of an inactivated whole-virus COVID-19 vaccine (VLA2001) compared with the adenoviral vector vaccine ChAdOx1-S in adults in the UK (COV-COMPARE): interim analysis of a randomised, controlled, phase 3, immunobridging trial.

BACKGROUND: The Valneva COVID-19 vaccine (VLA2001; Valneva Austria, Vienna, Austria) is an inactivated whole-virus, adjuvanted SARS-CoV-2 vaccine. We aimed to assess the safety and immunogenicity of primary vaccination with VLA2001 versus the ChAdOx1-S (Oxford-AstraZeneca) adenoviral-vectored vaccine. METHODS: In this immunobridging phase 3 trial (COV-COMPARE), participants aged 18 years and older who were medically stable (as determined by an investigator) were enrolled at 26 sites in the UK. In the double-blind, randomised, controlled arm of the trial, participants aged 30 years and older were randomly assigned (2:1) to receive two doses of VLA2001 (0·5 mL; with 33 antigen units [AU] per dose) or ChAdOx1-S (0·5 mL; with 2·5 × 108 infectious units per dose) on days 1 and 29. In another arm, participants aged 18-29 years received two doses of VLA2001 (same dose) open label on days 1 and 29. The primary immunogenicity outcome was the immune response of a two-dose schedule of VLA2001 on day 43, in adults aged 30 years and older, versus two doses of ChAdOx1-S via superiority of geometric mean titres (GMTs) of neutralising antibodies (GMT ratio of >1 at a two-sided significance level of 5%) and non-inferiority of the seroconversion rate (non-inferiority margin of -10% for the lower limit of the 95% CI for the difference between groups). The primary safety outcome was the frequency and severity of any adverse events in all participants up to day 43. Safety was assessed in all participants who received at least one dose of vaccine. GMTs were assessed in a subset of participants aged 30 years and older who were seronegative at baseline, had at least one evaluable antibody titre measurement after vaccination, and had no confirmed COVID-19 during the study (immunogenicity population); and seroconversion was assessed in the per-protocol population, which comprised the immunogenicity population but excluding any participants with major protocol violations. For each timepoint, only participants with available data were included in the analysis. This study is registered with ClinicalTrials.gov, NCT04864561, and is ongoing. FINDINGS: Between April 28 and June 3, 2021, 4181 individuals were screened and 4017 enrolled, of whom 2975 (74%) were aged 30 years or older and randomly assigned to receive VLA2001 (n=1978) or ChAdOx1-S (n=997), and 1042 (26%) were aged 18-29 years (all received open-label VLA2001). 4012 participants received at least one dose of vaccine (1040 in the open-label VLA2001 group, 1977 in the randomised VLA2001 group, and 995 in the ChAdOx1-S group). The immunogenicity population comprised 492 participants in the randomised VLA2001 group and 498 in the ChAdOx1-S group; three participants in the VLA2001 group were excluded from the per-protocol population. VLA2001 induced higher neutralising GMTs than did ChAdOx1-S (803·5 [95% CI 748·5-862·6] vs 576·6 [543·6-611·7]; GMT ratio 1·39 [95% CI 1·25-1·56]; p<0·0001), and non-inferior seroconversion rates (444 [97·4%] of 456 participants vs 444 [98·9%] of 449; difference -1·5% [95% CI -3·3 to 0·2]. Any adverse event was reported in 963 (92·6%) participants in the open-label VLA2001 group, 1755 (88·8%) in the randomised VLA2001 group, and 976 (98·1%) in the ChAdOx1-S group. Most adverse events reported were mild or moderate in severity. INTERPRETATION: VLA2001 has a favourable tolerability profile and met superiority criteria for neutralising antibodies and non-inferiority criterion for seroconversion rates compared with ChAdOx1-S. The data presented here formed the basis of successful marketing approval for use of VLA2001 in primary vaccination in the EU, the UK, Bahrain, and United Arab Emirates. FUNDING: UK Department of Health and Social Care and Valneva Austria.

Safety and immunogenicity of the inactivated whole-virus adjuvanted COVID-19 vaccine VLA2001: A randomized, dose escalation, double-blind phase 1/2 clinical trial in healthy adults.

OBJECTIVES: We aimed to evaluate the safety and optimal dose of a novel inactivated whole-virus adjuvanted vaccine against SARS-CoV-2: VLA2001. METHODS: We conducted an open-label, dose-escalation study followed by a double-blind randomized trial using low, medium and high doses of VLA2001 (1:1:1). The primary safety outcome was the frequency and severity of solicited local and systemic reactions within 7 days after vaccination. The primary immunogenicity outcome was the geometric mean titre (GMT) of neutralizing antibodies against SARS-CoV-2 two weeks after the second vaccination. The study is registered as NCT04671017. RESULTS: Between December 16, 2020, and June 3, 2021, 153 healthy adults aged 18-55 years were recruited in the UK. Overall, 81.7% of the participants reported a solicited AE, with injection site tenderness (58.2%) and headache (46.4%) being the most frequent. Only 2 participants reported a severe solicited event. Up to day 106, 131 (85.6%) participants had reported any AE. All observed incidents were transient and non-life threatening in nature. Immunogenicity measured at 2 weeks after completion of the two-dose priming schedule, showed significantly higher GMTs of SARS-CoV-2 neutralizing antibody titres in the highest dose group (GMT 545.6; 95% CI: 428.1, 695.4) which were similar to a panel of convalescent sera (GMT 526.9; 95% CI: 336.5, 825.1). Seroconversion rates of neutralizing antibodies were also significantly higher in the high-dose group (>90%) compared to the other dose groups. In the high dose group, antigen-specific IFN-γ expressing T-cells reactive against the S, M and N proteins were observed in 76, 36 and 49%, respectively. CONCLUSIONS: VLA2001 was well tolerated in all tested dose groups, and no safety signal of concern was identified. The highest dose group showed statistically significantly stronger immunogenicity with similar tolerability and safety, and was selected for phase 3 clinical development.

A trans-complementing recombination trap demonstrates a low propensity of flaviviruses for intermolecular recombination.

Intermolecular recombination between the genomes of closely related RNA viruses can result in the emergence of novel strains with altered pathogenic potential and antigenicity. Although recombination between flavivirus genomes has never been demonstrated experimentally, the potential risk of generating undesirable recombinants has nevertheless been a matter of concern and controversy with respect to the development of live flavivirus vaccines. As an experimental system for investigating the ability of flavivirus genomes to recombine, we developed a "recombination trap," which was designed to allow the products of rare recombination events to be selected and amplified. To do this, we established reciprocal packaging systems consisting of pairs of self-replicating subgenomic RNAs (replicons) derived from tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) that could complement each other in trans and thus be propagated together in cell culture over multiple passages. Any infectious viruses with intact, full-length genomes that were generated by recombination of the two replicons would be selected and enriched by end point dilution passage, as was demonstrated in a spiking experiment in which a small amount of wild-type virus was mixed with the packaged replicons. Using the recombination trap and the JEV system, we detected two aberrant recombination events, both of which yielded unnatural genomes containing duplications. Infectious clones of both of these genomes yielded viruses with impaired growth properties. Despite the fact that the replicon pairs shared approximately 600 nucleotides of identical sequence where a precise homologous crossover event would have yielded a wild-type genome, this was not observed in any of these systems, and the TBEV and WNV systems did not yield any viable recombinant genomes at all. Our results show that intergenomic recombination can occur in the structural region of flaviviruses but that its frequency appears to be very low and that therefore it probably does not represent a major risk in the use of live, attenuated flavivirus vaccines.

Helices alpha2 and alpha3 of West Nile virus capsid protein are dispensable for assembly of infectious virions.

The internal hydrophobic sequence within the flaviviral capsid protein (protein C) plays an important role in the assembly of infectious virions. Here, this sequence was analyzed in a West Nile virus lineage I isolate (crow V76/1). An infectious cDNA clone was constructed and used to introduce deletions into the internal hydrophobic domain which comprises helix alpha2 and part of the loop intervening helices alpha2 and alpha3. In total, nine capsid deletion mutants (4 to 14 amino acids long) were constructed and tested for virus viability. Some of the short deletions did not significantly affect growth in cell culture, whereas larger deletions removing almost the entire hydrophobic region significantly impaired viral growth. Efficient growth of the majority of mutants could, however, be restored by the acquisition of second-site mutations. In most cases, these resuscitating mutations were point mutations within protein C changing individual amino acids into more hydrophobic residues, reminiscent of what had been observed previously for another flavivirus, tick-borne encephalitis virus. However, we also identified viable spontaneous pseudorevertants with more than one-third of the capsid protein removed, i.e., 36 or 37 of a total of 105 residues, including all of helix alpha3 and a hydrophilic segment connecting alpha3 and alpha4. These large deletions are predicted to induce formation of large, predominantly hydrophobic fusion helices which may substitute for the loss of the internal hydrophobic domain, underlining the unrivaled structural and functional flexibility of protein C.

Neutralizing antibody persistence in pediatric travelers from non-JE-endemic countries following vaccination with IXIARO® Japanese encephalitis vaccine: An uncontrolled, open-label phase 3 follow-up study.

BACKGROUND: In an initial study among children from non-Japanese encephalitis (JE)-endemic countries, seroprotection rates remained high 6 months after completion of the primary series with IXIARO®. METHODS: In this open-label follow-up study, a subset of 23 children who received a 2-dose primary series of IXIARO® in the parent study, were evaluated for safety and neutralizing antibody persistence for 36 months. RESULTS: Seroprotection rates (SPRs) remained high but declined from 100% one month after primary immunization to 91.3% at month 7 and 89.5% at month 36. Geometric mean titers (GMTs) declined considerably from 384.1 by day 56-60.8 at month 36. No long-term safety concerns were identified. CONCLUSIONS: The substantial decline in GMT observed in this study, together with previously published data on children vaccinated with IXIARO® support the recommendation for a booster dose in children who remain at risk of JE from 1 year after the primary series of IXIARO®, consistent with the recommendation for adults. CLINICAL TRIAL REGISTRY NUMBER: NCT01246479.

Persistence of the immune response after vaccination with the Japanese encephalitis vaccine, IXIARO® in healthy adults: A five year follow-up study.

Immunization with the Japanese encephalitis (JE) vaccine IXIARO® results in protective neutralizing antibody levels for one year. Since persistence of protective titer levels beyond one year was unknown, a 5 years follow-up study was conducted. Additionally, data were stratified to compare the persistence of protective neutralizing antibodies against JE in people with or without tick-borne encephalitis (TBE) vaccination. Four weeks after the primary series, the percentage of subjects with PRNT50 titer ≥1:10 in the intent-to-treat population was 99%; the rate after 5 years was 81.6%. By month 24, 36, 48 and 60, the percentages were still 90.7%, 91.7%, 90.1%, 85.9%, respectively in the population who had received TBE vaccine compared to 67.9%, 71.9%, 69.1%, 63.8% in the population who had not. No long-term safety concerns were identified. These data indicate that vaccination with IXIARO® is able to induce protective titers that persist up to 60 months after the primary immunization. Clinical trial registry number NCT00596102.

Antibody Persistence up to 3 Years After Primary Immunization With Inactivated Japanese Encephalitis Vaccine IXIARO in Philippine Children and Effect of a Booster Dose.

BACKGROUND: An inactivated Vero cell culture derived Japanese encephalitis virus vaccine (IXIARO) requires a booster dose 1 year after primary schedule for long-term antibody persistence in adults. The aim of this study is to evaluate immunogenicity and safety of a booster dose in children 2 months to <18 years of age. METHODS: This is a randomized, controlled open-label study in the Philippines. Three hundred children vaccinated with IXIARO in a previous trial were randomized 1:1 to receive either no booster or a booster 12 months after initiation of the primary series. Neutralizing antibody titers were assessed before and after the booster and up to 3 years after primary series. Safety endpoints included the rate of subjects with solicited adverse events (AEs), unsolicited AEs and serious AEs within 1 month after the booster. RESULTS: Geometric mean titer declined by 1 year after the primary series, but titers remained above the established protective threshold in 85%-100% of children depending on age group. The booster led to a pronounced increase in geometric mean titer and 100% seroprotection rate in all age groups. The booster was well tolerated, with AE rates lower compared with the primary series. Most AEs were mild. CONCLUSIONS: A booster dose of IXIARO administered 12 months after the primary immunization was well tolerated and highly immunogenic.

Safety and immunogenicity of an inactivated Vero cell_derived Japanese encephalitis vaccine (IXIARO®, JESPECT®) in a pediatric population in JE non-endemic countries: An uncontrolled, open-label phase 3 study.

BACKGROUND: Young travelers to South-East Asia may be at risk for Japanese encephalitis (JE). METHODS: IXIARO® (0.25 ml or 0.5 ml, depending on age) were administrated to 100 travelers aged ≥ 2 months to < 18 years. Solicited AEs were collected for 7 days after each injection, unsolicited adverse events (AEs) for a total of 7 months. JE neutralizing antibodies were assessed in 64 subjects. RESULTS: The most common solicited local AEs were redness (3/12 subjects), induration and tenderness (both 1/12) with 0.25 ml IXIARO®, and tenderness (44/88) and pain (22/88) with 0.5 ml IXIARO®. Common solicited systemic AEs were diarrhea (2/12) and loss of appetite (1/12) with 0.25 ml IXIARO® and muscle pain (27/88) and excessive fatigue (10/88) with 0.5 ml IXIARO®. In total, up to day 56, AEs were reported by 10/12 (83.3%) of subjects who received the 0.25 ml dose and 67/88 (76.1%) of those vaccinated with the 0.5 ml dose. All subjects (62/62; 100%) developed protective levels of JE neutralizing antibodies by Day 56 and 31/34 (91.2%) retained protective titers at Month 7. CONCLUSIONS: IXIARO® was generally well tolerated in children, with an overall AE profile similar to adults. IXIARO® was highly immunogenic in both dose groups.

Characterization of West Nile virus live vaccine candidates attenuated by capsid deletion mutations.

Protein C deletion mutants of West Nile virus (WNV) were evaluated for their potential use as live virus vaccine candidates in vivo. Double and triple mutants carrying small deletions and second-site point mutations, as well as mutants with large deletions of 36 and 37 amino acid residues were tested in a stringent mouse challenge model. The mutant viruses were found to be non-pathogenic and to induce protective immunity in a wide range of inoculation doses (10(1)-10(6)FFU). Furthermore, the effects of combining three different previously identified resuscitating point mutations, as well as the combination of a large protein C deletion with a deletion mutation in the 3' non-coding region were studied. The data indicate that the production of safe and efficacious WNV live vaccines based on protein C deletion mutations is feasible.