Intracerebral transfection of anti-rabies virus antibodies is an effective therapy for rabies

Rabies is a neurological disease with 100% lethality. Some of the rare human patients who survived after multiple drug treatment had severe sequelae. The present study showed that after 48 h of RABV inoculation, mice injected intracerebrally with anti-RABV F (ab’)2 plus Bioporter® showed 70% survival compared to the control group, suggesting that transfection of anti-RABV antibodies to the brain may prevent or delay the spread of RABV at an early stage of infection. This result may provide important protocol results in intracellular antibody delivery to prevent the fatal outcome of the disease.


Introduction
Rabies is a zoonotic neurological disease with 100% lethality; some of the few human patients who survived after a multidrug treatment developed severe motor impairment due to ischemic encephalopathy followed by necrosis of the hippocampus, cerebellum, and cortex (Burton et al. 2005;Manoj et al. 2016). The use of immunomodulators and antivirals has not been shown to be effective in inhibiting the progression of the disease when tested in mice and humans (van Thiel et al. 2009;Marosi et al. 2018). Circa 59,000 human deaths occur worldwide yearly due to rabies, and although it is preventable with pre and post-exposure prophylaxis, the logistics and costs involved in rabies treatments are a limiting factor to saving lives (Hampson et al. 2015).
Rabies lyssavirus RABV (Mononegavirales:Rhabdoviridae: Lyssavirus) is a neurotropic virus with a circa 11 kb negativesense single-stranded RNA as a genome that codes for the nucleoprotein (N), phosphoprotein (P), matrix protein (M), envelope glycoprotein (G), and the RNA-dependent RNA-polymerase L protein (Jackson and Wunner 2007), and it is most often transmitted amongst mammals via saliva after an initial local replication in muscle cells that follows to the central nervous system (CNS) via axons (Lewis et al. 2000). Within a variable period of time after infection, signs of hyperactivity, hypersalivation and hydrophobia are detectable. The virus causes enough damage to the brain in a few days that the infection invariably leads to coma and death by cardio-respiratory arrest (WHO 2013).
Here, we show that the use of intracerebral transfection of anti-RABV antibodies to treat mice inoculated with RABV reduces mortality and extends the incubation period of rabies.

Results
Probing the transfection to mouse brains with Bioporter® complexed with an FITC-antibody control protein (Genlantis) after intracerebral inoculations in mice showed fluorescent foci at 4-6 h post-injection, in brain slices obtained in a cryomicrotome (Fig. 1), evidencing the efficacy of the protein transfection to mouse brains. However, the fluorescence technique performed with microscopic slide tissue fragments showed absence of fluorescent foci using an anti-equine mouse IgG conjugate for the mice injected with the F (ab') 2 anti-RABV Bioporter® complex at concentrations of 50 and 250 μg/mL for post-injection periods of 4-6 h.
The survival rate in the Group treated with Bioporter® plus anti-RABV F (ab') 2 was as high as 70% with a minimum Washington C. Agostinho and Paulo E. Brandão contributed equally to this work. incubation period of 7 days, resulting in a significant difference (p < 0.00198) tested by Fischer's exact test when compared to the Control group. On the other hand, mice which received anti-RABV F (ab') 2 without Bioporter® (Control group) had onset of rabies symptoms at 5 d.p.i. Ninety percent of the mice showed symptoms in 2 to 3 days, followed by death, with only one mouse showing no symptoms throughout the experiment, resulting in 10% of survival. The clinical signs observed were anorexia, piloerection, arching of the back, and limb paralysis before death (Fig. 2).
Bioporter® alone had no significant action on RABV, as morbidity/mortality rates of 50% and 80% were found for mice treated with only Bioporter® or Hepes solution, respectively, after 48 h of RABV inoculation (p = 0.3498) tested by Fischer exact test, indicating that the reduced morbidity/ mortality rate in mice treated with anti-RABV F (ab') 2 transfected with Bioporter was due to a specific intracellular neuralization effect (Table 1).
All dead animals were positive for direct immunofluorescence for RABV, with no difference in fluorescence intensity between groups. Thirty days after viral inoculation, all surviving mice were euthanized and negative by direct immunofluorescence and PCR for RABV.

Discussion
After the treatment performed with Bioporter® 48 h after RABV inoculation, 70% of the mice in the treated group were healthy after viral infection, suggesting intense inhibition of the virus by anti-RABV F (ab') 2 . The anti-RABV F (ab') 2 was transfected using a single intracerebral inoculation with optimal result. These results are consistent with previous observations showing inhibition of viral activity in N2A cell culture inoculated with isolates BOV-AgV3 (IP4005/10), DOG-AgV2 (IP3629/11), 964/06-Eptesicus furinalis, and Pasteur virus (PV) (Kawai 2012). Therefore, transfection of anti-RABV F (ab') 2 in vivo, demonstrated in this study could, act in other variants with the same result confirmed for DOG-AgV2 (IP3629/11), especially in cases of human rabies.
The possibility of the Bioporter® reagent contributing to the antiviral action together with the anti-RABV F (ab') 2 required further investigation. When this was tested, Bioporter® was inoculated into mice in the CNS without addition of anti-RABV F (ab') 2 , and the acute phase of morbidity and mortality in mice occurred between the 5th and 10th d.p.i. with 80% mortality in the control groups, demonstrating that the inhibitory effect of treatment was due only to the neutralizing action of anti-RABV F (ab') 2 . Though not significant, if this inhibitory capacity of Bioporter® is due to a lytic effect on virions or to the sequestration of viral proteins needed for virion assembly remains to be investigated.
Some infections in the CNS are contained by the action of several immune effectors such as antibodies, cytotoxic Tcells, and soluble factors that are involved in generation and control of the immune response as type 1 IFNs. Consequently, after brain infection by a pathogen, MHC II expression is upregulated by approximately 90% of glial cells, including in diffuse areas distal to viral infection (Hooper et al. 2009). In murine brain, infection demonstrates the prolonged The serological status of rabies patients show that the production of antibodies plays a fundamental role in viral clearance. Complete recovery with or without sequels in rabies patients is limited to a few cases in the literature linked to the history of immunization with the combination of vaccines and passive immunization of antibodies at the onset of symptoms (Willoughby et al. 2005;PROMED 2009).
The IFD technique with equine anti-IgG conjugate to detect transfection of anti-RABV F (ab') 2 by Bioporter reagent performed 4-6 h after injection showed no transfected antibody. Lack of visualization of intracellular F (ab') 2 does not necessarily imply non-intracytoplasmic presence and may indicate that (a) transfection of anti-RABV F (ab') 2 occurred with low efficiency; (b) its intracytoplasmic dispersion avoids large clusters of anti-RABV F (ab') 2 accumulation, in this way the fluorescence of the anti-IgG conjugate is inhibited by fluorescence microscopy if it is associated with anti-RABV F (ab') 2 ; and (c) the result may be related to lack of affinity of the equine anti-IgG conjugate by the fragmentation of IgG-RABV.
Interestingly, in the work done by Weiil et al. (Weill et al. 2008), among the three antibodies transfected in vitro by the Pulsin® reagent, the only one that did not demonstrate the expected signaling was the anti-mouse IgG antibody, since a secondary antibody rapidly exuded from the cytoplasm when the cells were treated with digitonin (lipid solubilizer) revealing that it was not bound to any target, while the primary antibodies remained within the cytoplasm for 15 min. This result helps to support hypothesis (b) in which the dispersion of anti-RABV F (ab') 2 in the cytoplasm, implies nonvisualization of its location by signaling by the equine anti-IgG conjugate.
A fluorescence microscopy depends on detection of the fluorophore above its effective detection limit, and false negatives may occur when attempting to study the dispersion of fluorophore-labeled molecules, so that a low level of fluorescence could reflect the absence in that tissue area or a high degree of dispersion would decrease the fluorescence to the point of not being distinguished of the autofluorescence of the tissue attached to the slide (Ho et al. 2018).
The data shown herein suggest that transfection of anti-RABV antibodies is a new candidate tool to aid in the treatment of rabies, when no post-exposure prophylaxis has been applied or when prophylaxis has failed.
In order to gather further evidence of the efficiency of this treatment and to overcome some of the limitations of the experiments described herein, the use of a more diverse set of RABV strains that includes CVS-11, which, at the adequate doses, would give a 100% lethality in mice and should provide a better dose-effect measure of the antiviral effect, though some fixed stain might lose the pathogenic properties of their ancestors (Davis et al. 2015), and bat-related strains, after adaptation no mouse CNS as a model, is needed. It is also of note that the low lethality observed in the mice might be a result of a low RABV dosis (10 3.8 LD50/30 μL) and increasing this dosis is mandatory for a better assessment of the effects observed herein, as higher titer could produce an advantage for the virus in the CNS, increasing the mortality rate in mice (Fuoco et al. 2018). Also, increasing the number of tested mice could provide a more clear understanding on the interference of factors other than RABV F (ab') 2 on the survival rates.

Transfection test with Bioporter® protein delivery reagent in vivo
First, in order to assess the effectivity of protein delivery to mice brains, a total of 40 μl of FITC-antibody control protein (Gelantis) was complexed with the Bioporter® Protein Delivery Reagent (Genlantis) as per the manufacturer's Table 1 Survival of mice inoculated intracerebrally with 10 3.8 LD50/ 30 μL RABV DOG-IP3629/11 and treated 48 h, post-inoculation treated group (Bioporter + anti-RABV F (ab') 2 , control groups (anti-RABV F (ab') 2 + Hepes) and (Bioporter + Hepes), with comparison by Fisher's exact test. The p value with the result significant at p < 0.05
Transfection test with anti-RABV F (ab') 2 in vivo Next, 20 female 21-day-old CH3 ROCKEFELLER mice were intracerebrally inoculated with RABV strain IP3629/11-AgV2 isolated from a dog in Brazil in mouse central nervous system (CNS) with a previously determined titer in mice CNS of 10 3.8 LD50/ 30 μL, kindly provided by the Pasteur Institute, Brazil. Females were chosen instead of a mixed population as (a) it is known that the susceptibility to viral infection differs from females to males (Barna et al. 1996;Muller et al. 1995) and (b) females are easier to handle than males. A dog-related RABV strain was used instead of the fixed reference strain CVS-11 because dogs are still the main reservoirs for human rabies worldwide, and this choice could more promptly provide data on the efficacy of the treatment against street strains of RABV. After 48 h, mice were intracerebrally injected with 40 μL of either Bioporter® resuspended in 20 mM Hepes pH 7.4 containing anti-RABV F (ab') 2 (treated group, n = 10 mice) or Hepes 20 mM pH 7.4 containing anti-RABV F (ab') 2 (control group, n = 10 mice), both groups with 0.17 UI (250 μg) of anti-RABV F (ab') 2 as a final dose.

Evaluation of the action of the transfection agent alone on RABV
To assess whether the Bioporter transfection agent alone had any effects on RABV, 20 female 21-day-old CH3 ROCKEFELLER mice were intracerebrally inoculated with RABV strain IP3629/11-AgV2 and the test group (n = 10 mice) was injected with 40 μL of Bioporter in 400 μl Hepes 20 mM pH 7.4, and the control group (n = 10 mice), was injected with 40 μL of Hepes 20 mM pH 7.4 solution, all after 48 h of RABV inoculation.

Equine anti-IgG conjugate
For evaluation of transfection of equine F (ab') 2 against RABV by direct immunofluorescence, mouse anti-horse IgG (whole molecule) -FITC antibody (Sigma-Aldrich) was used.

Virus
Strain (IP3629/11-AgV2 dog isolated from Brazil), grown on mice central nervous system (CNS) with a title of 10 3.8 LD50/ 30 μL for mice CNS, kindly provided by the Pasteur Institute, Brazil, was used for the infection in mice. This RABV variant was chosen because dogs are still the main transmitters of rabies worldwide.

Antibody transfection test
FITC-antibody control protein (Gelantis) complexed with Bioporter® Protein Delivery Reagent (Genlantis) per manufacturer's instructions were injected by the intracerebral route in two mice, and the CNS of each mouse was collected at 4 or 6 h post-injection; 10 μm sections were obtained in a LEICA CM 1860 UV microtoime, fixed in glass sliced with − 20°C acetone/2 h and observed for fluorescence with a OLYMPUS ® BX53 epifluorescence microscope.

DFAT and PCR
All mice were observed for a period 30 days after RABV inoculation for signs of rabies such as anorexia, piloerection hyperesthesia, aggressiveness, paralysis, and death, being the surviving mice euthanized at the end of the observation period. The central nervous system (CNS) of each mouse was tested with a direct fluorescent antibody test (DFAT), using an anti-RABV nucleocapsid IgG conjugates with fluorescein isothiocyanate (Pasteur Institute, Brazil), and, if negative, to a PCR targeting RABV N-P genes.

Statistical analysis
GraphPad Prism was used for statistical analyses of in vivo data using Fisher's exact test with α = 0.05.

Funding information
The authors are thankful for the financial support provided by CNPq (Brazilian National Board for Scientific and Technological Development) grant number 307291/2017-0 and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil) Finance Code 001, which had no role in the study design, collection, analysis and interpretation of data, writing of the report, and in the decision to submit the article for publication.