Immunogenicity and Safety of a Purified Vero Rabies Vaccine - Serum Free, Compared With Two Licensed Vaccines, in a Simulated Rabies Post-Exposure Regimen in Healthy Adults in France: A Randomized Controlled Phase III Trial.

BACKGROUND: A next-generation Vero cell rabies vaccine (PVRV-NG2) was developed using the same Pitman-Moore strain as in the licensed purified Vero cell vaccine (PVRV; Verorab®) and the human diploid cell vaccine (HDCV; Imovax Rabies®). METHODS: This dual-center, modified double-blind, phase III study in France evaluated immunogenic non-inferiority and safety of PVRV-NG2 with and without concomitant intramuscular human rabies immunoglobulin (HRIG), compared with PVRV+HRIG and HDCV+HRIG, in a simulated post-exposure prophylaxis (PEP) regimen. Healthy adults ≥18 years old (N=640) were randomized 3:1:1:1 to receive PVRV-NG2+HRIG, PVRV+HRIG, HDCV+HRIG, or PVRV-NG2 alone (administered as single vaccine injections on days [D] 0, 3, 7, 14, and 28, with HRIG administered on D0 in applicable groups). Rabies virus neutralizing antibodies (RVNA titers) were assessed pre- (D0) and post-vaccination (D14, D28, and D42) using the rapid fluorescent focus inhibition test. Non-inferiority, based on the proportion of participants achieving RVNA titers ≥0.5 IU/mL (primary objective), was demonstrated if the lower limit of the 95% CI of the difference in proportions between PVRV-NG2+HRIG and PVRV+HRIG/HDCV+HRIG was >-5% at D28. Safety was assessed up to 6 months after the last injection. RESULTS: The non-inferiority of PVRV-NG2+HRIG, compared with PVRV+HRIG and HDCV+HRIG, was demonstrated. Nearly all participants (99.6%, PVRV-NG2+HRIG; 100%, PVRV+HRIG; 98.7%, HDCV+HRIG; 100%, PVRV-NG2 alone) achieved RVNA titers ≥0.5 IU/mL at D28. Geometric mean titers were similar between groups with concomitant HRIG administration at all time points. Safety profiles were similar between PVRV-NG2 and comparator vaccines. CONCLUSIONS: In a simulated PEP setting, PVRV-NG2+HRIG showed comparable immunogenicity and safety to current standard-of-care vaccines. CLINICAL TRIALS REGISTRATION: NCT03965962.

Safety and immunogenicity of a serum-free purified Vero rabies vaccine in comparison with the rabies human diploid cell vaccine (HDCV; Imovax® Rabies) administered in a simulated rabies post-exposure regimen in healthy adults.

A new generation, serum-free, antibiotic-free, purified Vero rabies vaccine (PVRV-NG; Sanofi) has been developed based on the same Pitman-Moore viral strain used for the currently licensed purified Vero cell rabies vaccine (PVRV; Verorab®, Sanofi) and human diploid cell vaccine (HDCV; Imovax® Rabies, Sanofi). PVRV-NG has demonstrated a satisfactory safety profile and induces robust immune responses, with non-inferiority demonstrated versus PVRV when given as a three-dose pre-exposure prophylaxis (PrEP) regimen in healthy children and adults. Here, we evaluated the safety and immunogenic non-inferiority of PVRV-NG compared to HDCV when administered as simulated post-exposure prophylaxis (PEP), with concomitant administration of human rabies immunoglobulin (HRIG), in healthy adults in the USA. Participants were vaccinated according to the 5-dose Essen intramuscular regimen (4-week, 1-injection site regimen, with a single dose given on days 0, 3, 7, 14 and 28) for PEP, with concomitant HRIG administered on day 0. Rabies virus neutralising antibodies (RVNA) were evaluated on days 0, 14, 28 and 42. Non-inferiority of PVRV-NG compared with HDCV was shown if the lower limit of the 95 % confidence interval (CI) for the difference in seroconversion rates (RVNA titers ≥ 0.5 IU/mL on day 14) between PVRV-NG and HDCV was above the non-inferiority margin of -5 %. Safety was evaluated after each vaccination and monitored throughout the study. The difference in seroconversion rate between the PVRV-NG and HDCV groups was -2.8 % (95 % CI, -8.08 to 4.20), indicating that non-inferiority was not demonstrated. The seroconversion rate was < 99 % in both study groups on day 14. There were no major safety concerns identified, and PVRV-NG demonstrated a similar safety profile to HDCV.

Safety and immunogenicity of three dose levels of an investigational, highly purified Vero cell rabies vaccine: A randomized, controlled, observer-blinded, Phase II study with a simulated post-exposure regimen in healthy adults.

A serum-free, highly purified rabies vaccine produced in Vero cells is under development. The initial formulation, PVRV-NG, was evaluated in five Phase II studies and subsequently reformulated (PVRV-NG2). This multicenter, observer-blinded Phase II study investigated the safety and immune response of three different doses (antigen content) of PVRV-NG2 versus a licensed human diploid cell rabies vaccine (HDCV; Imovax rabies®). Healthy adults (N = 320) were randomized to receive PVRV-NG2 (low, medium, or high dose), PVRV-NG, or HDCV (2:2:2:1:1 ratio), according to a five-dose Essen simulated post-exposure regimen (Days [D] 0, 3, 7, 14, and 28). All participants received human rabies immunoglobulin intramuscularly on D0. Immunogenicity was assessed at D0, 14, 28, 42, and 6 months after the final injection using the rapid fluorescent focus inhibition test. Seroconversion rates were calculated as the percentage of participants achieving rabies virus neutralizing antibody titers ≥0.5 IU/mL. All analyses were descriptive. At each timepoint, geometric mean titers (GMTs) increased with antigen content (measured using an enzyme-linked immunosorbent assay). High-dose PVRV-NG2 GMTs were the highest at all timepoints, medium-dose PVRV-NG2 GMTs were similar to those with HDCV, and low-dose PVRV-NG2 GMTs were similar to PVRV-NG. The safety profile of PVRV-NG2 was comparable to PVRV-NG; however, fewer injection site reactions were reported with PVRV-NG2 or PVRV-NG (range 36.7-47.5%) than with HDCV (61.5%). This study demonstrated a dose-effect of antigen content at all timepoints. As post-exposure prophylaxis, the safety and immunogenicity profiles of the high-dose PVRV-NG2 group compared favorably with HDCV. Clinicaltrials.gov number: NCT03145766.

Safety and immunogenicity of a serum-free purified Vero rabies vaccine in healthy adults: A randomised phase II pre-exposure prophylaxis study.

A serum-free, highly purified Vero cell rabies vaccine (PVRV-NG) is under development. We previously demonstrated that pre-exposure prophylaxis (PrEP) with PVRV-NG had a satisfactory safety profile and was immunogenically non-inferior to the licensed purified Vero cell rabies vaccine in adults. Here, we evaluated the safety and immunogenic non-inferiority of PrEP with PVRV-NG compared to the licensed human diploid cell vaccine (HDCV) in healthy adults (NCT01784874). Participants received three vaccinations (days 0, 7, and 28) as PrEP with or without a booster injection after 12 months. Rabies virus neutralising antibodies (RVNA) were evaluated on days 0, 28 (subgroup only), and 42, and Months 6, 12, and 12 + 14 days (booster group only). Non-inferiority (first primary objective) was based on the proportion of participants with RVNA titres ≥ 0.5 IU/mL (World Health Organization criteria for seroconversion) on day 42, expected to be ≥ 99% (second primary objective). Safety was evaluated after each dose and monitored throughout the study. At day 42, PVRV-NG was non-inferior to HDCV and the first primary objective was met; seroconversion was observed for 98.3% of PVRV-NG recipients and 99.1% of HDCV recipients. As < 99% of participants in the PVRV-NG group had RVNA titres ≥ 0.5 IU/mL, the second primary objective was not met. Booster vaccination produced a strong increase in RVNA titres for all groups, primed with PVRV-NG or HDCV. RVNA geometric mean titres tended to be higher for HDCV than PVRV-NG primary vaccine recipients. In a complementary evaluation using alternative criteria for seroconversion (complete virus neutralization at 1:5 serum dilution), 99.6% and 100% of participants in the PVRV-NG and HDCV groups, respectively, achieved seroconversion across the vaccine groups. No major safety concerns were observed during the study. PVRV-NG was well tolerated, with a similar safety profile to HDCV in terms of incidence, duration, and severity of adverse events after primary and booster vaccinations. ClinicalTrials.gov number: NCT01784874.

Serum-free purified Vero rabies vaccine is safe and immunogenic in children: Results of a randomized phaseII pre-exposure prophylaxis regimen study.

BACKGROUND: A serum-free, highly purified Vero rabies vaccine (PVRV-NG) has been developed with no animal or human components and low residual DNA content. A phaseII randomized clinical study aimed to demonstrate the non-inferiority of the immune response and assess the safety profile of PVRV-NG versus a licensed human diploid cell culture rabies vaccine (HDCV) in a pre-exposure regimen in healthy children and adolescents in the Philippines. METHODOLOGY: Children aged 2-11 years and adolescents aged 12-17 years were randomized (2:1) to receive three injections of either PVRV-NG or HDCV (on day [D] 0, D7 and D28). Rabies virus-neutralizing antibodies (RVNA) were measured at D0, D42 and 6 months after the first injection (month [M] 6). Safety was assessed during the vaccination period and up to 28 days after the last vaccination. Serious adverse events were followed until 6 months after last vaccination. PRINCIPAL FINDINGS: 342 healthy participants (171 children and 171 adolescents) were randomized and followed for 6 months after the last dose. All participants in both groups had an RVNA titer ≥ 0.5 IU/ml at D42, demonstrating non-inferiority in seroconversion rate for PVRV-NG versus HDCV. Over 90% of participants had RVNA titer ≥ 0.5 IU/ml at M6. PVRV-NG was well tolerated after each vaccination and up to 6 months following the last dose. There were no major safety concerns during the study, and the type and severity of solicited adverse events was similar for both treatment groups. CONCLUSIONS: This study demonstrated the non-inferior immune profile of PVRV-NG compared with HDCV in a pre-exposure setting within a pediatric population. PVRV-NG was well tolerated with no safety concerns. This study is registered at ClinicalTrials.gov (NCT01930357) and EU Clinical Trials Register (2015-003203-30).

Inhibitory effect of concomitantly administered rabies immunoglobulins on the immunogenicity of commercial and candidate human rabies vaccines in hamsters.

The World Health Organization protocol for rabies post-exposure prophylaxis (PEP) recommends extensive wound washing, immediate vaccination, and administration of rabies immunoglobulin (RIG) in severe category III exposures. Some studies have shown that RIG can interfere with rabies vaccine immunogenicity to some extent. We investigated the interference of RIG on a next generation highly purified Vero cell rabies vaccine candidate (PVRV-NG) versus standard-of-care vaccines in a previously described hamster model. The interference of either human (h) or equine (e) RIG on the immune response elicited by PVRV-NG, Verorab® (purified Vero cell rabies vaccine, PVRV), and Imovax® Rabies (human diploid cell rabies vaccine; HDCV) was evaluated using the 4-dose Essen PEP regimen. The anti-rabies seroneutralizing titers and specific serum IgM titers were measured by fluorescent antibody virus neutralization test and enzyme-linked immunosorbent assay, respectively, for the vaccines administered with or without RIG. The RIG interference on PVRV-NG, observed transiently at Day 7, was similar to that on PVRV and tended to be lower than that on HDCV using both read-outs. In summary, the results generated in the hamster model showed that RIG induced similar or less interference on PVRV-NG than the standard-of-care vaccines.

Potency test to discriminate between differentially over-inactivated rabies vaccines: Agreement between the NIH assay and a G-protein based ELISA.

The NIH assay is used to assess the potency of rabies vaccine and is currently a key measure required for vaccine release. As this test involves immunization of mice and subsequent viral challenge, efforts are being made to develop alternative analytical methods that do not rely on animal testing. Sanofi Pasteur has reported the development of a G-protein specific ELISA assay that has shown agreement with the NIH test. In this study we have generated several non-conform vaccine lots by an excessive inactivation with ß-propiolactone (BPL) and assessed the capacity of both tests to detect the corresponding consequences. Excessive BPL inactivation causes G-protein unfolding, altering in turn viral morphology and the continuity of the G-protein layer in the viral particle. Both the NIH and the ELISA tests were able to monitor the consequences of excessive inactivation in a similar manner. Of note, the experimental error of the ELISA test was well below that of the NIH test. These results increase the prospect that the ELISA test could be considered a suitable candidate for the replacement of the NIH test.

Biophysical virus particle specific characterization to sharpen the definition of virus stability.

Vaccine thermostability is key to successful global immunization programs as it may have a significant impact on the continuous cold-chain maintenance logistics, as well as affect vaccine potency. Modern biological and biophysical techniques were combined to in-depth characterize the thermostability of a formulated rabies virus (RABV) in terms of antigenic and genomic titer, virus particle count and aggregation state. Tunable resistive pulse sensing (TRPS) and nanoparticle tracking analysis (NTA) were used to count virus particles while simultaneously determining their size distribution. RABV antigenicity was assessed by NTA using a monoclonal antibody that recognize a rabies glycoprotein (G protein) conformational epitope, enabling to specifically count antigenic rabies viruses. Agreement between antigenicity results from NTA and conventional method, as ELISA, was demonstrated. Additionally, NTA and ELISA showed mirrored loss of RABV antigenicity during forced degradation studies performed between 5 °C and 45 °C temperature exposure for one month. Concomitant with decreased antigenicity, emergence of RABV particle populations larger than those expected for rabies family viruses was observed, suggesting RABV aggregation induced by thermal stress. Finally, using a kinetic-based modeling approach to explore forced degradation antigenicity data (NTA, ELISA), a two-step model accurately describing antigenicity loss was identified. This model predicted a RABV shelf-life of more than 3 years at 5 °C; significant loss of antigenicity was predicted for samples maintained several months at ambient temperature. This thorough characterization of RABV forced degradation study originally provided a time-temperature mapping of RABV stability.

G-protein based ELISA as a potency test for rabies vaccines.

The NIH test is currently used to assess the potency of rabies vaccine, a key criterion for vaccine release. This test is based on mice immunization followed by intracerebral viral challenge. As part of global efforts to reduce animal experimentation and in the framework of the development of Sanofi Pasteur next generation, highly-purified vaccine, produced without any material of human or animal origin, we developed an ELISA as an alternative to the NIH test. This ELISA is based on monoclonal antibodies recognizing specifically the native form of the viral G-protein, the major antigen that induces neutralizing antibody response to rabies virus. We show here that our ELISA is able to distinguish between potent and different types of sub-potent vaccine lots. Satisfactory agreement was observed between the ELISA and the NIH test in the determination of the vaccine titer and their capacity to discern conform from non-conform batches. Our ELISA meets the criteria for a stability-indicating assay and has been successfully used to develop the new generation of rabies vaccine candidates. After an EPAA international pre-collaborative study, this ELISA was selected as the assay of choice for the EDQM collaborative study aimed at replacing the rabies vaccine NIH in vivo potency test.

Replacement of in vivo human rabies vaccine potency testing by in vitro glycoprotein quantification using ELISA - Results of an international collaborative study.

Three different ELISAs quantifying rabies glycoprotein were evaluated as in vitro alternatives to the National Institutes of Health (NIH) in vivo potency test for batch release of human rabies vaccines. The evaluation was carried out as an international collaborative study supported by the European Partnership for Alternatives to Animal Testing (EPAA). This pre-validation study, the results of which are presented in this paper, compared three different ELISA designs, assessing their within- and between-laboratory precision. One of the ELISA designs was proposed to the European Directorate for the Quality of Medicines & HealthCare (EDQM) and accepted for an international collaborative study under the umbrella of the Biological Standardisation Programme.

Study of rabies virus by Differential Scanning Calorimetry.

Differential Scanning Calorimetry (DSC) has been used in the past to study the thermal unfolding of many different viruses. Here we present the first DSC analysis of rabies virus. We show that non-inactivated, purified rabies virus unfolds cooperatively in two events centered at approximately 62 and 73 °C. Beta-propiolactone (BPL) treatment does not alter significantly viral unfolding behavior, indicating that viral inactivation does not alter protein structure significantly. The first unfolding event was absent in bromelain treated samples, causing an elimination of the G-protein ectodomain, suggesting that this event corresponds to G-protein unfolding. This hypothesis was confirmed by the observation that this first event was shifted to higher temperatures in the presence of three monoclonal, G-protein specific antibodies. We show that dithiothreitol treatment of the virus abolishes the first unfolding event, indicating that the reduction of G-protein disulfide bonds causes dramatic alterations to protein structure. Inactivated virus samples heated up to 70 °C also showed abolished recognition of conformational G-protein specific antibodies by Surface Plasmon Resonance analysis. The sharpness of unfolding transitions and the low standard deviations of the Tm values as derived from multiple analysis offers the possibility of using this analytical tool for efficient monitoring of the vaccine production process and lot to lot consistency.

A next-generation, serum-free, highly purified Vero cell rabies vaccine is safe and as immunogenic as the reference vaccine Verorab® when administered according to a post-exposure regimen in healthy children and adults in China.

BACKGROUND: As an evolution of its currently licensed rabies vaccine Verorab(®), Sanofi Pasteur has developed a next-generation, serum-free, highly purified Vero rabies vaccine (PVRV-NG). Through this Phase III clinical trial, we aimed to demonstrate the non-inferiority of PVRV-NG over Verorab when administered according to a post-exposure regimen and to assess its clinical safety. METHODS: A total of 816 healthy subjects aged ≥10 years were randomized according to a 2:1 ratio to receive PVRV-NG or Verorab. Half of the subjects were aged 10-17 years, the other half were aged ≥18 years. All subjects were to receive 5 injections on days 0, 3, 7, 14 and 28. Three blood samples were taken for rabies virus neutralizing antibodies (RVNA) assessment, at baseline, on day 14 and day 42. Solicited adverse reactions (between injections 1, 2 and 3, and within 7 days post-injections 4 and 5) and adverse events (up to 28 days after the last injection) were collected for clinical safety assessment; serious adverse events were reported up to 6-months after the last injection. RESULTS: The proportion of subjects with an RVNA titer ≥0.5 IU/mL after the third injection of PVRV-NG was non-inferior to the proportion of those who received Verorab. PVRV-NG was shown to be as immunogenic as Verorab in each age range in the per-protocol and full analysis sets. PVRV-NG induced a strong immune response in both age ranges, with high RVNA levels and increased geometric mean titers compared to baseline after each measured time point. PVRV-NG had a satisfactory safety profile after each injection, similar to Verorab with regards to the nature, frequency, duration and severity of adverse events. Two serious adverse events were reported, none was related to vaccination. CONCLUSIONS: This trial demonstrated the immunogenic non-inferiority of PVRV-NG over Verorab and showed that both vaccines have similar safety profiles. This trial is registered at ClinicalTrials.gov (NCT01339312). This manuscript is the first full report of the study. An abstract of the study results was previously presented at the Rabies in the Americas (RITA) conference in October 2012 in São Paulo, Brazil. FUNDING: Sanofi Pasteur.

A serum-free, purified vero cell rabies vaccine is safe and as immunogenic as the reference vaccine Verorab for pre-exposure use in healthy adults: results from a randomized controlled phase-II trial.

BACKGROUND: Verorab was licensed in 1985 for both pre- and post-exposure prophylaxis of rabies. The next generation purified Vero cell rabies vaccine (PVRV-NG) is a highly purified vaccine. We performed a phase II clinical study in adults in France to assess its immunological non-inferiority and clinical safety for pre-exposure prophylaxis. METHODS: In a randomized phase-II trial, 384 healthy adult subjects were randomized (2:1) to receive a three-dose primary series of PVRV-NG or Verorab. One year later, the PVRV-NG group received a PVRV-NG booster while the Verorab group participants were randomized to receive a booster of PVRV-NG or Verorab for. Rabies virus neutralizing antibodies (RVNA) were evaluated on days 0, 28 (subgroup), 42, months 6, 12 and 12+14 days. Safety was evaluated for seven days after each dose. Adverse event between doses, until 28 days after the final dose was recorded. Serious adverse events were recorded up to 6 months after the last dose. RESULTS: The criterion for non-inferiority was met in the per-protocol analysis set and confirmed in the full analysis set (FAS). In the FAS, 99.6% and 100% of subjects had RVNA titers ≥0.5 IU/mL in PVRV-NG and Verorab groups, respectively. While RVNA levels gradually decreased over the 12-month period, at 6 and 12 months after vaccination >89% and >77%, respectively, in both groups had RVNA titers ≥0.5 IU/mL. The PVRV-NG booster induced a strong response, irrespective of the vaccine given for the primary series. PVRV-NG was safe and well tolerated and its safety profile was similar to Verorab for unsolicited adverse events and solicited systemic reactions. The incidence of solicited injection-site reactions was lower with PVRV-NG than with Verorab after the primary series and the booster dose. CONCLUSIONS: PVRV-NG was shown to be at least as immunogenic as Verorab and to present a similar safety profile.

Transferrin-binding protein B of Neisseria meningitidis: sequence-based identification of the transferrin-Binding site confirmed by site-directed mutagenesis.

A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6. Site-directed mutagenesis of residues located in these domains has led to the identification of two domains, amino acids 53 to 57 and 240 to 245, which are involved in binding to human transferrin (htf). These two domains are conserved in an alignment of different TbpB sequences from N. meningitidis and Neisseria gonorrhoeae, indicating a general functional role of the domains. Western blot analysis and BIAcore and isothermal titration calorimetry experiments demonstrated that site-directed mutations in both binding domains led to a decrease or abolition of htf binding. Analysis of mutated proteins by circular dichroism did not provide any evidence for structural alterations due to the amino acid replacements. The TbpB mutant R243N was devoid of any htf-binding activity, and antibodies elicited by the mutant showed strong bactericidal activity against the homologous strain, as well as against several heterologous tbpB isotype I strains.