Anti-rabies Monoclonal Antibodies Research Articles

A phase 2b, Randomized, double blinded comparison of the safety and efficacy of the monoclonal antibody mixture SYN023 and human rabies immune globulin in patients exposed to rabies

BackgroundSYN023 is an anti-rabies monoclonal antibody mixture administered as part of post-exposure prophylaxis regimens. The rabies virus neutralizing antibody (RVNA) concentration generally accepted as an adequate immune response to vaccination is ≥ 0.5 IU/mL. MethodsWithin 54 h of potential rabies exposure, 448 patients in two risk substrata of WHO Category III exposure were randomized to receive either 0.3 mg/kg SYN023 or 0.133 mL/kg human rabies immunoglobulin (HRIG) injected in and around the wound site(s) plus a course of rabies vaccination. Patients were followed for safety and absence of rabies for ≥ 365 days. ResultsGMT RVNA was higher with SYN023 throughout the 2-week post-treatment period. In the primary analysis group (n = 368), 99.4 % of SYN023 recipients versus 4.5 % of HRIG recipients had protective RVNA levels on Day 4. On Day 8, 98.1 % SYN023 versus 12.2 % HRIG recipients were protected. The SYN023:HRIG ratio of geometric mean titer of RVNA (RVNA GMTs) on Day 8 (19.42) exceeded the 10 % superiority margin (P < 0.0001) indicating higher Day 8 RVNA with SYN023. On Day 99, the SYN023:HRIG RVNA GMT ratio (0.66) was below the non-inferiority margin of 20 % (P = 0.9485) suggesting some moderation of vaccine immune response by SYN023 relative to HRIG. The ratio of percent SYN023:HRIG recipients achieving RVNA ≥ 0.5 IU/mL on Day 99 (0.98) met the non-inferiority margin of 20 % (P = 0.013) indicating anti-rabies immune response with SYN023 was non-inferior to HRIG despite this effect. There were no probable/confirmed rabies cases in any patient. Study regimens were well tolerated. ConclusionsSYN023 provided higher RVNA than HRIG soon after rabies exposure. By Day 99 post-treatment, GM RVNA with SYN023 was lower than HRIG, however, the percent of SYN023 recipients with a protective response was not inferior at this time point. No rabies cases were reported in the study. The SYN023 safety profile was acceptable.ClinicalTrials.gov ID: NCT03961555.

Comparing recombinant human rabies monoclonal antibody (ormutivimab) with human rabies immunoglobulin (HRIG) for postexposure prophylaxis: A phase III, randomized, double-blind, non-inferiority trial

ObjectivesTo evaluate the immunogenicity and safety of an anti-rabies monoclonal antibody (mAb), ormutivimab, compared with human rabies immunoglobulin (HRIG). MethodsThis phase III trial was designed as a randomized, double-blind, non-inferiority clinical trial in patients aged ≥18 years with suspected World Health Organization category Ⅲ rabies exposure. The participants were randomized 1:1 to ormutivimab and HRIG groups. After thorough wound washing and injection of ormutivimab/HRIG on day 0, the vaccination was administered on days 0, 3, 7, 14, and 28. The primary endpoint was the adjusted geometric mean concentration (GMC) of rabies virus-neutralizing activity (RVNA) on day 7. The endpoint of safety included the occurrence of adverse reactions and serious adverse events. ResultsA total of 720 participants were recruited. The adjusted-GMC of RVNA (0.41 IU/ml) on day 7 in ormutivimab group was not inferior to that in the HRIG group (0.41 IU/ml), with ratio of adjusted-GMC of 1.01 (95% confidence interval: 0.91, 1.14). The seroconversion rate of the ormutivimab group was higher than that of the HRIG group on days 7, 14, and 42. Most local injection sites and systemic adverse reactions reported from both groups were mild to moderate in severity. Conclusionormutivimab + vaccine can protect victims aged ≥18 years with category Ⅲ suspected rabies exposure as a component of postexposure prophylaxis. ormutivimab has a weaker influence on the immunity response of rabies vaccines. Clinical trials registrationChiCTR1900021478 (the Chinese Clinical Trial Registry of World Health Organization).

Epitope mapping of diagnostic anti-rabies monoclonal antibodies provide structural insight into rabies variant identification in infected wildlife.

Rabies virus is a member of the lyssavirus family and causes over 60,000 deaths a year. There are currently five strains of rabies virus circulating throughout the continental United States. In order to better control the spread of rabies virus, the Center of Disease Control monitors the prevalence and host range of different rabies virus variants (RVV) throughout the United States. This surveillance relies on a panel of monoclonal antibodies that are known to differentially bind to the nucleoprotein of RVV in a direct fluorescent antibody test (DFA). While these tests are known to accurately distinguish RVV, it is unknown where these antibodies bind to the rabies nucleoprotein. To better understand the fundamental principles of this assay, the epitopes of three monoclonal antibodies from the current panel, which are suspected to have conformational epitopes, were determined using hydrogen-deuterium mass spectrometry. We determined these antibodies bind to distinct surface-exposed epitopes of the nucleoprotein, one of which makes contacts with multiple subunits of the 11-mer ring that is formed by the nucleoprotein. After determining the epitopes, a multiple sequence alignment was examined to look at the conservation of the amino acids in each epitope to determine how these antibodies distinguish between RVV. By determining epitopes, we can now predict how viral mutations could change the specificity and/or sensitivity of a rabies DFA. The results of this work can be used to help further develop rapid immunoassays for more economical RVV diagnostics in the field, in regions that may lack access to microscopy-based DFA diagnostics.

Population Pharmacodynamic Analyses of Human Anti-Rabies Virus Monoclonal Antibody (Ormutivimab) in Healthy Adult Subjects.

Ormutivimab is the first recombinant human anti-rabies monoclonal antibody (rhRIG) approved for clinical application in China. In this study, a population pharmacodynamic (PPD) model was established to compare the neutralizing antibody activities of Ormutivimab and human rabies immunoglobulin (HRIG), alone or combined with human rabies vaccine (Vero), in a phase II clinical trial, and to recommend a target dose for the phase III trial. The model was verified to fit the PPD data well. The stability of the model was verified by the bootstrap method. The level of neutralizing antibodies in vivo increased rapidly after administration of Ormutivimab or HRIG. Neutralizing antibodies with a strong activity were produced at 7 days (Ormutivimab + vaccine) or 10 days (HRIG + vaccine) after induction by the vaccine in vivo. Compared to that induced by HRIG + vaccine, the level of the neutralizing antibodies induced by Ormutivimab + vaccine peaked higher and faster. The levels of neutralizing antibodies induced by Ormutivimab + vaccine and HRIG + vaccine were similar within 21 days after administration. According to these results and the safety data, 20 IU·kg−1 was recommended as the target dose in the confirmatory study of Ormutivimab. Registration: ClinicalTrials.gov #NCT02559921.

Efficacy of ormutivimab, a novel recombinant human anti-rabies monoclonal antibody, in post-exposure prophylaxis animal models

Human rabies is a serious public health problem that can't be ignored. Rabies immune globulin (RIG) is an indispensable component of rabies post-exposure prophylaxis (PEP). However, current PEP relies on RIG purified from pooled human or equine plasma, which are either in chronic shortage or associated with safety concerns. Monoclonal antibodies have become widely accepted as safer and more cost-effective alternatives to RIG products in recent years. Here, we assessed the neutralization breadth of human monoclonal antibody ormutivimab and its protective efficacy in PEP models. Ormutivimab was able to neutralize a broad panel of Chinese prevalent street RABVs with neutralizing potency form 198–1487.6 IU/mL. Furthermore, ormutivimab offered comparable protection to that with HRIG both at standard doses (20 IU/kg) and higher doses (100 IU/kg and 200 IU/kg). The interference of ormutivimab on vaccine potency was also analyzed and found slightly reduced neutralizing antibody titers similar to HRIG. The broad-spectrum neutralization activities, highly protective potency, and rapid onset of action make ormutivimab an effective candidate for human rabies PEP.

Effect of an Endoplasmic Reticulum Retention Signal Tagged to Human Anti-Rabies mAb SO57 on Its Expression in Arabidopsis and Plant Growth.

Transgenic Arabidopsis thaliana expressing an anti-rabies monoclonal antibody (mAb), SO57, was obtained using Agrobacterium-mediated floral dip transformation. The endoplasmic reticulum (ER) retention signal Lys-Asp-Glu-Leu (KDEL) was tagged to the C-terminus of the anti-rabies mAb heavy chain to localize the mAb to the ER and enhance its accumulation. When the inaccurately folded proteins accumulated in the ER exceed its storage capacity, it results in stress that can affect plant development and growth. We generated T1 transformants and obtained homozygous T3 seeds from transgenic Arabidopsis to investigate the effect of KDEL on plant growth. The germination rate did not significantly differ between plants expressing mAb SO57 without KDEL (SO plant) and mAb SO57 with KDEL (SOK plant). The primary roots of SOK agar media grown plants were slightly shorter than those of SO plants. Transcriptomic analysis showed that expression of all 11 ER stress-related genes were not significantly changed in SOK plants relative to SO plants. SOK plants showed approximately three-fold higher mAb expression levels than those of SO plants. Consequently, the purified mAb amount per unit of SOK plant biomass was approximately three times higher than that of SO plants. A neutralization assay revealed that both plants exhibited efficient rapid fluorescent focus inhibition test values against the rabies virus relative to commercially available human rabies immunoglobulins. KDEL did not upregulate ER stress-related genes; therefore, the enhanced production of the mAb did not affect plant growth. Thus, KDEL fusion is recommended for enhancing mAb production in plant systems.

A Phase 3, Randomized, Open-label, Noninferiority Trial Evaluating Anti-Rabies Monoclonal Antibody Cocktail (TwinrabTM) Against Human Rabies Immunoglobulin (HRIG).

Limited supply, cost and potential for severe adverse effects observed with the blood derived rabies immunoglobulin products has led to search for alternative therapies. This issue has been addressed by developing an anti-rabies monoclonal antibody cocktail. This is a phase 3, randomized, open-label, noninferiority trial conducted in patients with World Health Organization (WHO) category III exposure with suspected rabid animal. Eligible patients were assigned to either the test arm, TwinrabTM (docaravimab and miromavimab) or the reference arm, human rabies immunoglobulin (HRIG; Imogam® Rabies-HT), in a ratio of 1:1. The primary endpoint was the comparison of responder rates between the 2 arms assessed as percentage of those with rabies virus neutralizing antibodies titers ≥0.5 IU/mL on day 14. A total of 308 patients were equally randomized into the 2 arms. In the per-protocol (PP) population, there were 90.21% responders in the TwinrabTM arm and 94.37% in the HRIG arm. The geometric mean of rapid fluorescent foci inhibition test titers in the PP on day 14 were 4.38 and 4.85 IU/mL, for the TwinrabTM and HRIG arms, respectively. There were no deaths or serious adverse events reported. This study confirmed that TwinrabTM is noninferior to HRIG in terms of providing an unbroken window of protection up to day 84. This trial in healthy adults with WHO category III exposure from suspected rabid animal also establishes the safety of TwinrabTM in patients with 1 WHO approved vaccine regimen (Essen). CTRI/2017/07/009038.

In Vivo Efficacy of SYN023, an Anti-Rabies Monoclonal Antibody Cocktail, in Post-Exposure Prophylaxis Animal Models.

Rabies immune globulin (RIG) is an indispensable component of rabies post-exposure prophylaxis (PEP) because it provides passive immunity to prevent this otherwise inescapably fatal disease in Category III exposed patients. Even with decades of development, RIG products are still criticized for their high cost, lot-to-lot variation, and potential safety issues. They remain largely unattainable in most developing regions of the world, where demand is highest. In recent years, monoclonal antibodies (MAbs) have become widely accepted as safer and more cost-effective alternatives to RIG products. As an example, SYN023 is a 1:1 cocktail of two humanized anti-rabies MAbs previously shown to display extensive neutralizing capabilities. Here, we further assessed the efficacy of SYN023 in animal models of rabies, and found that SYN023 afforded protection equal to a standard dose of human RIG (HRIG) at 0.03 mg/kg in Syrian hamsters and 0.1 mg/kg in beagles. Potential interference with vaccine-induced immunity was analyzed for the MAbs at these concentrations. While individual MAbs did not interfere with vaccine response, SYN023 at dosages of 0.1 mg/kg and above resulted in reduced neutralizing antibody titers similar to HRIG. Thus, the in vivo characterization of SYN023 supports its utility in human rabies PEP as an efficacious alternative to RIG products.

A Phase 3, Randomised, Open-Label, Non-Inferiority Trial Evaluating Anti-Rabies Monoclonal Antibody Cocktail (Twinrab &lt;sup&gt;TM&lt;/sup&gt;) Against Human Rabies Immunoglobulin (HRIG)

Background: Rabies is a zoonotic disease caused by RNA (ribonucleic acid) viruses of the genus, Lyssavirus of the family, Rhabdoviridae. Limited supply, cost and potential for severe adverse effects observed with the blood derived rabies immunoglobulin products has led to the search for alternative therapies. To address this issue, Cadila Healthcare Ltd. in collaboration with the World Health Organization (WHO) has developed an anti-rabies monoclonal antibody cocktail based product. Methods: This was a phase 3, randomized, multi-centric, open-label, comparator-controlled, non-inferiority trial conducted in 308 patients with WHO category III suspected rabies exposures. Patients meeting the eligibility criteria were assigned to one of the two treatment arms, TwinrabTM or Human rabies immunoglobulin (HRIG; Imogam® Rabies-HT), in a ratio of 1:1. The study drug was administered to patients on Day 0. Five doses of rabies vaccine were also administered (Day 0, 3, 7, 14 and 28) to all the patients following the WHO Essen Schedule. The primary endpoint was the difference in responders [assessed as Rapid Fluorescent Focus Inhibition Test (RFFIT) titre ≥0·5 IU/mL on Day 14] in the TwinrabTM arm as compared to the HRIG arm. Findings: Three hundred and eight patients were randomized; 130 (90·28%) patients in the TwinrabTM arm and 134 (94·37%) patients in the HRIG arm had an RFFIT titre ≥0·5 IU/ml in the per-protocol (PP) population. The Geometric Mean of RFFIT titre in the same population on Day 14 was 4·38 and 4·85 IU/mL respectively, in the TwinrabTM and the HRIG arms. In the modified intent to treat (mITT) population on Day 14, there were 133 (89·86%) responders in the TwinrabTM arm and 137 (90·73%) responders in the HRIG arm. There were no deaths or any other serious adverse events reported during the study period in any of the treatment arms. Interpretation: This study confirmed that TwinrabTM is non-inferior to HRIG in terms of providing an unbroken window of protection as assessed by RFFIT titers up to Day 84. This trial also establishes the safety of TwinrabTM in patients with WHO category III suspected rabies exposures. Trial Registration: CTRI/2017/07/009038. Funding Statement: This trial was sponsored and funded by CHL., Ahmedabad, India. Declaration of Interests: All authors declared no competing interests. Ethics Approval Statement: Written informed consent was given by all study subjects and by parents/legal guardians in cases of children. The consent process was audio-visually recorded at the time of screening for all subjects, as per the local regulations. This trial was initiated after obtaining the approvals of Ethics Committees (ECs), Drugs Controller General of India (DCGI) (dated 17 March 2017) and registering the trial with the Clinical Trial Registry of India (CTRI). This trial was conducted in accordance with the applicable local regulations.

The study of degradation mechanisms of glyco-engineered plant produced anti-rabies monoclonal antibodies E559 and 62-71-3.

Rabies is an ancient and neglected zoonotic disease caused by the rabies virus, a neurotropic RNA virus that belongs to the Rhabdoviridae family, genus Lyssavirus. It remains an important public health problem as there are cost and health concerns imposed by the current human post exposure prophylaxis therapy. The use of monoclonal antibodies (mAbs) is therefore an attractive alternative. Rabies mostly affects people that reside in resource-limited areas where there are occasional failures in the cold-chain. These environmental changes may upset the stability of the mAbs. This study focused on mAbs 62-71-3 and E559; their structures, responses to freeze/thaw (F/T) and exposure to reactive oxygen species were therefore studied with the aid of a wide range of biophysical and in silico techniques in order to elucidate their stability and identify aggregation prone regions. E559 was found to be less stable than 62-71-3. The complementarity determining regions (CDR) contributed the most to its instability, more specifically: peptides 99EIWD102 and 92ATSPYT97 found in CDR3, Trp33 found in CDR1 and the oxidised Met34. The constant region “158SWNSGALTGHTFPAVL175” was also flagged by the special aggregation propensity (SAP) tool and F/T experiments to be highly prone to aggregation. The E559 peptides “4LQESGSVL11 from the heavy chain and 4LTQSPSSL11 from the light chain, were also highly affected by F/T. These residues may serve as good candidates for mutation, in the aim to bring forward more stable therapeutic antibodies, thus paving a way to a more safe and efficacious antibody-based cocktail treatment against rabies.

Recent advances in the development of monoclonal antibodies for rabies post exposure prophylaxis: A review of the current status of the clinical development pipeline

Despite successful control in many parts of the world, rabies virus continues to result in tens of thousands of deaths each year. Death from rabies can be prevented by timely and appropriate post exposure prophylaxis including wound cleaning and administration of vaccine and rabies immunoglobulin. Currently, rabies immunoglobulin is derived from the blood plasma of horses or humans and has several limitations relating to supply, cost and quality. Monoclonal antibodies produced through recombinant DNA technologies could potentially overcome these limitations. The first anti-rabies monoclonal antibody has recently gained regulatory approval in India and there are several other candidates being evaluated in clinical trials. Given the advances in the field, rabies monoclonal antibodies have been recently considered by the World Health Organization’s Strategic Advisory Group of Experts on Immunization and included in updated WHO immunization policy recommendations for rabies published in April 2018. This article reviews the current landscape of the clinical trial development of anti-rabies monoclonal antibodies and the historical clinical trial pathways followed for blood-derived rabies immunoglobulin before discussing challenges in the clinical evaluation, regulatory approval, uptake and monitoring of these products.

Generation and Characterization of a Neutralizing Monoclonal Antibody Against Rabies Virus

Rabies is a zoonotic disease, for which effective treatment methods after the onset of clinical symptoms have not been developed yet. Polyclonal sera, both human and equine, along with vaccines are important means of disease prophylaxis. However, due to adverse reactions to the immunoglobulins of animal origin, high cost, and limited availability of the safer human serum, polyclonal antibodies should be substituted for a stable and efficient preparation, which is recombinant neutralizing antirabies monoclonal antibodies (mAbs). This paper reports generation of the humanized mAb 1C5, which binds with the antigenic site (AS) III of the rabies virus glycoprotein (RABVG) and demonstrates high virus neutralization activity in the fluorescent antibody virus neutralization test, as a result of expression in the Chinese hamster ovary (CHO) cells.

Expression, glycosylation, and function of an anti-rabies virus monoclonal antibody in tobacco and Arabidopsis plants

Plants have emerged as one of the most attractive systems for producing human therapeutic proteins against viral diseases. These include diagnostic reagents, vaccines, and antibodies. This process is known as molecular biofarming. The objective of this study was to develop and evaluate tobacco and Arabidopsis as plant platforms for producing human anti-rabies monoclonal antibody (mAb). Both tobacco and Arabidopsis transgenic plants were generated by Agrobacterium-mediated transformation. Purification of mAb SO57K from each plant was performed with ammonium sulfate-mediated precipitation and protein A affinity columns. SDS–PAGE analysis showed that the purity of mAb SO57K obtained from each transgenic plant was similar, whereas Arabidopsis showed approximately twofold greater protein expression than tobacco. The N-glycosylation was not significantly different between proteins from the two plant species, with both showing oligo-mannose glycan structures. The mAbs SO57 derived from both the model plants had similar neutralizing efficacy against target virus strain CVS-11. Taken together, tobacco and Arabidopsis are both promising platforms for producing a human anti-rabies mAb.

The Lyssavirus glycoprotein: A key to cross-immunity

Rabies is an acute viral encephalomyelitis in warm-blooded vertebrates, caused by viruses belonging to Rhabdovirus family and genus Lyssavirus. Although rabies is categorised as a neglected disease, the rabies virus (RABV) is the most studied amongst Lyssaviruses which show nearly identical infection patterns. In efforts to improving post-exposure prophylaxis, several anti-rabies monoclonal antibodies (mAbs) targeting the glycoprotein (G protein) sites I, II, III and G5 have been characterized. To explore cross-neutralization capacity of available mAbs and discover new possible B-cell epitopes, we have analyzed all available glycoprotein sequences from Lyssaviruses with a focus on sequence variation and conservation. This information was mapped on the structure of a representative G protein. We proposed several possible cross-neutralizing B-cell epitopes (GUVTTTF, WLRTV, REECLD and EHLVVEEL) in complement to the already well-characterized antigenic sites. The research could facilitate development of novel cross-reactive mAbs against RABV and even more broad, against possibly all Lyssavirus members.

Efficient In Vitro and In Vivo Activity of Glyco-Engineered Plant-Produced Rabies Monoclonal Antibodies E559 and 62-71-3.

Rabies is a neglected zoonotic disease that has no effective treatment after onset of illness. However the disease can be prevented effectively by prompt administration of post exposure prophylaxis which includes administration of passive immunizing antibodies (Rabies Immune Globulin, RIG). Currently, human RIG suffers from many restrictions including limited availability, batch-to batch inconsistencies and potential for contamination with blood-borne pathogens. Anti-rabies monoclonal antibodies (mAbs) have been identified as a promising alternative to RIG. Here, we applied a plant-based transient expression system to achieve rapid, high level production and efficacy of the two highly potent anti-rabies mAbs E559 and 62-71-3. Expression levels of up to 490 mg/kg of recombinant mAbs were obtained in Nicotiana benthamiana glycosylation mutants by using a viral based transient expression system. The plant-made E559 and 62-71-3, carrying human-type fucose-free N-glycans, assembled properly and were structurally sound as determined by mass spectrometry and calorimetric density measurements. Both mAbs efficiently neutralised diverse rabies virus variants in vitro. Importantly, E559 and 62-71-3 exhibited enhanced protection against rabies virus compared to human RIG in a hamster model post-exposure challenge trial. Collectively, our results provide the basis for the development of a multi-mAb based alternative to RIG.

Commentary: Incorporation of membrane-anchored flagellin or Escherichia coli heat-labile enterotoxin B subunit enhances the immunogenicity of rabies virus-like particles in mice and dogs.

Rabies virus is accountable for substantial economic burden, and possesses high risk to the individuals living in low-income countries. Rabies is fatal encephalitis caused by the rabies virus (RABV) and usually transmit to humans through bites from rabid animals. The causative agent for this life threatening disease is RABV, containing single-stranded negative-sense RNA as genetic material (Jackson, 2013). Annually, rabies is responsible for approximately 55,000 deaths worldwide (a substantial part of deaths occur in developing countries); (Ge et al., 2011). Vaccination is the principal path for prevention and control of the disease. Currently available cell culture based rabies vaccines possess several side effects (e.g. fever, headache, neurological disease etc.) and high cost in developing countries usually require many doses to provide pre and post-exposure protection against the RABV. Several, improved vaccines based on anti-rabies monoclonal antibodies, lyophilized, and cell culture technology, are already under clinical development phase (Kaur et al., 2015). Furthermore, recombinant molecules have also been used as vaccine candidates very extensively. Further, immunogenicity of these molecules can be enhanced by combining adjuvants and immuno-stimulatory molecules. In the past, Wen et al. demonstrated that recombinant rabies virus (rRABV) expressing dendritic cell activating molecules such as granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage-derived chemokine (MDC), and macrophage inflammatory protein (MIP-1a), could enhance RABV immunogenicity (Wen et al., 2011). Moreover, Zhou et al. suggested that rRABV expressing bacterial flagellin could also induce robust adaptive immune responses (Zhou et al., 2013). Flagellin is a very effective and potent adjuvant because of non-inducer of IgE production and at very low dose (1–10 μg in non-human primates) very efficient to induce effective immune responses (Mizel and Bates, 2010). Recombinant virus-like particles (VLPs) are promising vaccine candidates, because they mimic original virus structure and are able to induce potent humoral and cellular immune responses (Kumar et al., 2011). Moreover, VLP possesses several advantages over the conventional vaccines because of the lack of genetic material, stable in extreme environment and capable of expressing foreign molecules. Very recently, Fontana et al. (2014) showed that VLPs containing glycoprotein of RABV are very efficient in induction of antibody mediated immune responses in Balb/c mice. Qi et al. (2015) combined all these novel strategies, in order to develop an effective RABV vaccine. In present report, Qi and colleagues constructed chimeric RABV-like particles (cRVLPs) containing RABV glycoprotein and matrix protein, expressing membrane-anchored flagellin or E. coli heat-labile enterotoxin B (LTB). Immunological studies in BALB/c mice and dog models demonstrated that both humoral and cellular immune responses were stronger with cRVLPs, compared with standard rabies VLPs (sVLPs). Further, Qi et al. (2015) suggest that flagellin promote antigen processing and presentation by inducing higher expression of MHC I and II on the surface of DCs. Interestingly, in a pioneer study, mucosal delivery of inactivated influenza vaccine have been shown to induce heterotypic immunity against H1N1 virus, when administered with heat-labile enterotoxin B (Tumpey et al., 2001). These attributes make flagellin and enterotoxin B outstanding adjuvants for use in vaccines. In conclusion, present and previous reports suggest that chimeric VLPs are novel class of subunit vaccines and possess capability to induce both protective innate and adaptive immune responses. Possibly, use of immuno-modulators along with cRVLPS may enhance the efficacy of existing rabies vaccines. Further, detailed mechanism of the comprehensive effect of flagellin and enterotoxin B stimulated T and B-cells, on antiviral immune responses, should be investigated.

Safety and pharmacokinetics of a human monoclonal antibody to rabies virus: A randomized, dose-escalation phase 1 study in adults

BackgroundRabies is an essentially fatal disease that is preventable with the timely administration of post-exposure prophylaxis (PEP). The high cost of PEP, which includes vaccine and hyperimmune globulin, is an impediment to the goal of preventing rabies in the developing world. Recently a recombinant human IgG1 anti-rabies monoclonal antibody (SII RMab) has been developed in India to replace serum-derived rabies immunoglobulin. The present study was conducted to demonstrate the safety of SII RMab and to determine the dose resulting in neutralizing serum antibody titers comparable to human rabies immunoglobulin (HRIG) when administered in conjunction with rabies vaccine in a simulated PEP regimen. MethodsThis randomized, open label, dose-escalation phase 1 study was conducted in healthy adults at a large tertiary care, referral, public hospital in India. Safety was assessed by active surveillance for adverse events along with standard laboratory evaluations and measurement of anti-drug antibodies (ADA). Anti-rabies antibody levels were measured by rapid fluorescent focus inhibition test (RFFIT) and ELISA. The study duration was 365 days. FindingsSII RMab was well tolerated with similar frequency of local injection site reactions to HRIG. The geometric mean concentrations of rabies neutralizing antibody in the vaccine plus SII RMab 10IU/kg cohort were comparable to the vaccine plus HRIG 20IU/kg cohort throughout the 365-day study period; day 14 geometric mean concentrations 23.4IU/ml (95% CI 14.3, 38.2) vs. 15.3IU/ml (95% CI 7.72, 30.3; p=NS), respectively. Future post-exposure prophylaxis studies of SII RMab at a dose of 10IU/kg in conjunction with vaccine are planned.

Antigenic and genetic characterization of rabies viruses isolated from domestic and wild animals of Brazil identifies the hoary fox as a rabies reservoir

Fifty Brazilian rabies viruses, collected from many different animal species and several regions of the country, were characterized by partial sequencing of the central, variable region of the P gene, a locus useful for sensitive molecular epidemiological studies. Phylogenetic analysis of the sequences, which included comparison with other rabies strains recovered from throughout the Americas, identified three main groups of Brazilian viruses, arbitrarily designated BRL-1 to BRL-3. BRL-1 was found in terrestrial carnivores and clusters with other American strains of the cosmopolitan lineage. BRL-2 comprised two distinct isolates, recovered from two species of non-haematophagous bats, that had evolutionary links to insectivorous-bat-derived strains of North America. BRL-3 consisted of isolates from vampire bats and from livestock species probably infected via contact with vampire bats. The terrestrial group was further subdivided into three subtypes: BRL-1a was associated exclusively with dogs and cats, while BRL-1b and BRL-1c were found exclusively in hoary foxes. These observations strongly support the role of the Brazilian hoary fox as a rabies reservoir. Screening of representative Brazilian rabies viruses against a collection of anti-rabies monoclonal antibodies (mAbs) identified a small panel of mAbs that could be used to discriminate between all Brazilian subgroups as defined by genetic classification in this study.

A recombinant human Fab expressed in Escherichia coli neutralizes rabies virus.

A recombinant human anti-rabies monoclonal antibody (MAb-57) Fab was prepared by cloning the heavy (Fd)- and light-chain domains into the same bacterial expression vector. To construct the recombinant Fab, mRNA was extracted from MAb-57-producing hybridoma cells, reverse transcribed, and then amplified by polymerase chain reaction (PCR) by using oligonucleotides specific for immunoglobulin heavy- and light-chain DNA sequences. PCR-amplified Fd-chain cDNA was fused, in frame, between a bacterial leader peptide (PelB) at the amino terminus and a 10-amino-acid peptide tag at the carboxy terminus. The PCR-amplified lambda-chain cDNA was also fused to the PelB leader peptide. The immunoglobulin Fab was then expressed as a dicistronic message in bacteria by using the isopropyl-beta-D-thiogalactopyranoside-inducible lactose promotor (lacZ). DNA sequencing was used to define the gamma-chain isotype (immunoglobulin G1) and VH (VHI) chain and VL (V lambda II) chain gene usage. The recombinant Fab (rFab57) specifically bound the rabies virus coat glycoprotein, while the Fd and lambda chains, when expressed individually, did not. The binding specificity of rFab57 was indistinguishable from that of the intact MAb in direct enzyme-linked immunosorbent assays; however, the dissociation constant of rFab57 for rabies virus protein G was approximately 1 log10 U lower than that of complete MAb-57 in competition enzyme-linked immunosorbent assays. A fluorescent-focus inhibition assay showed that bacterially expressed rFab was capable of neutralizing rabies virus strain CVS-11. We conclude that a human Fab expressed in bacteria maintains its specificity and biologic activity.

Inhibition of immune responses against rabies virus by monoclonal antibodies directed against rabies virus antigens

Treatment of mice with a cocktail of murine anti-rabies monoclonal antibodies (mAb-C) interfered with the ability of these animals to mount a virus-neutralizing antibody response to rabies vaccine. Administered mAb-C did not affect the induction of rabies virus-specific T-helper cells. The magnitude of the inhibition of rabies virus-specific B-cell response was dependent on the concentration of the mAb-C and the duration of the mAb-mediated interference was inversely proportional to the biological half-life of the mAb. As long as the serum titres were above a critical threshold, the suppression could not be overcome even by multiple vaccinations. Since injection of mice with immunocomplexes consisting of inactivated rabies virus and mAb rendered the animals non-responsive to a subsequent vaccination with inactivated rabies virus, it is concluded that the mAb-induced suppression might be caused by the formation of antigen-antibody complexes which exert a negative signalling effect to premature B cells.