rabies vaccines

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Presenting by :Nagendra P16PBT204

M.Tech Pharmaceutical Biotechnology

Department of Pharmaceutical Sciences and Technology

INSTITUTE OF CHEMICAL TECHNOLOGYMatunga, Mumbai

Rabies Vaccines : Prospects & Challenges

Under the guidance of :Prof. Vandana B Patravale

2 Rabies Rabies is a zoonotic disease which is caused by a virus. Rabies virus (RABV) belongs to the type species of the genus Lyssavirus

within the family Rhabdoviridae. Rabies infects domestic and wild animals, and is spread to people through

close contact with infected saliva through bite, scratch, aerosols etc., Dogs, Bats, wild cats, Jackals, wolves etc

3 Rabies Statistics (2015)

Deaths across the globe

IndiaRest of the world

INDIA

36%Deaths In Asia

India

Rest of Asia

INDIA

65%

http://www.who.int/rabies/epidemiology/en/

20,000

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Australia

New zealand

Japan

U.K.

IcelandNorway

5 Structure of Lyssavirus

Warrell, M. J., & Warrell, D. A. (2004). Rabies and other lyssavirus diseases. The Lancet, 363(9413), 959-969.

75 nm

300 nm

6 RNA Genome of Rabies Virus (RABV)12 kb

Baron, S. (1996). Rabdoviruses: RabiesVirus .In Medical Microbiology, 4th edition. Galveston, Texas.

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Schnell, M. J., McGettigan, J. P., Wirblich, C., & Papaneri, A. (2010). The cell biology of rabies virus: using stealth to reach the brain. Nature Reviews Microbiology, 8(1), 51-61.

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Baron, S. (1996). Rabdoviruses: RabiesVirus .In Medical Microbiology, 4th edition. Galveston, Texas.

9 Virus Infection

http://www.rsgplus.org/wp-content/uploads/2016/06/kas_ch232_f002.png

10RABV infection starts at a peripheral site (e.g., skin or muscle tissue).

The virus enters unmyelinated nerve terminals and migrates by retrograde axonal transport to the neuronal cell body.

After replication in the cell body of the primary neuron, infection proceeds via retrograde axonal transport and trans-synaptic spread through several neurons and reach the CNS.

Then it infects the acinar cells which releases the virus into the oral cavity by passing on to the salivary glands.

Dietzschold, B., Schnell, M., & Koprowski, H. (2005). Pathogenesis of rabies. In The World of Rhabdoviruses (pp. 45-56). Springer Berlin Heidelberg.

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12 Pathogenesis The incubation period varies from 2 weeks to 6 years depending on

the amount of virus in the inoculum and the site of inoculation First clinical symptom is usually neuropathic pain at the wound

site. Encephalitis Virus grows to high titers in the salivary glands Negri bodies appear in neuron cell bodies

Clinical spectrum Prodrome - nausea, headaches, fever, sore throat, photophobia Acute neurologic phase - nervousness, hallucinations, behavioral

anomalies, salivation, perspiration, hydrophobia, Coma and death.

http://peir.path.uab.edu/library/picture.php?/9055

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https://infogr.am/rabies-7022587310

14 Diagnosis

Based on the history Signs and symptoms Clinical examination Detection of antigen by taking skin biopsy using immunofluorescence. Virus isolation from saliva & other secretions. CSF analysis, MRI and CT scan. ELISA RT-PCR direct Fluorescent Antibody (DFA) testing Negri bodies

15 Direct fluorescent antibody test The ideal tissue to test for rabies antigen is brain because rabies is present in nervous

tissue (and not blood like many other viruses).

Flouresecent (fluorescein isothiocyanate) labeled antibody is incubated with rabies-suspect brain tissue.

If antigen present, it will bind.

Unbound antibody can be washed away.

Visualized under fluorescence microscope.

If rabies virus is absent there will be no staining.

http://www.cdc.gov/rabies/diagnosis/direct_fluorescent_antibody.html

16 Prevention and Treatment

Pre-Exposure Prophylaxis Administration of vaccines.

Post-Exposure Prophylaxis Administration of vaccines along with virus

neutralizing antibodies (VNA) such as Human Rabies Immuno globulins (HRIG) or Equine Rabies Immuno globulins.

http://www.sinosourcebio.com/products-d.php?id=48https://trade.indiamart.com/search.mp?search=rabies+immunoglobulin

17 Vaccines

Vaccines

Classical

Live Attenuated Inactivated

Synthetic

Peptides DNA

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http://www.historyofvaccines.org/content/rabies-treatment

19 History of Rabies Vaccine

1885

• Louis Pasteur developed the earliest effective vaccine (on 6 July 1885).• He used the homogenates of RABV infected rabbit spinal cord as innoculates.

1930’s

• Then they inactivated infected sheep or goat brain with chemical agents such as phenol and used them as vaccines.

• Alternatively, they also used inactivated infected chick embryos and infected suckling mouse brain.

1970’s

• Development of cell culture for virus propagation was initiated.• Growth of fixed RABV in a human diploid cell line such as MRC-5. These were termed

as Human Diploid Cell Vaccines (HDCV) .• An alternative to HDCV is the use of purified chick embryo cells (PCEC).

20 Inactivated rabies vaccines for humans

Cell Types Human diploid cells, Primary dog kidney, Vero, BHK-21, Primary chick embryo, Primary hamster kidney cells

and Primary chick embryo cells

Virus Strains Flury (LEP)(40-50 passages), Flury (HEP)(227-230), Pitmann-moore (PM), Evelyn rokitniki abelseth

(ERA), CVS-11 (kissling strain) and Paris pasteur strains of rabbit

fixed rabies virus

WHO recommended several approved rabies vaccine strains, adapted to various different cell types:

Sugiyama, M., & Ito, N. (2007). Control of rabies: epidemiology of rabies in Asia and development of new-generation vaccines for rabies. Comparative immunology, microbiology and infectious diseases, 30(5), 273-286

21 Administration of Rabies VaccinesPre-exposure vaccination Include for travellers travelling to rabies endemic regions,

veterinarians and researchers working with the virus. Consists of an intramuscular injection of 1 ml vaccine on days 0, 7,

21 and 28. boosting may be recommended at an interval of 3–5years

Post-exposure vaccination Typically given as an intra-muscular injection on days 0, 3, 7, 14

and 30. HRIG or ERIG against viral glycoprotein G is given on day 0, unless the recipient has received previous vaccination against rabies.

World Health Organization. (2005). WHO expert consultation on rabies: first report.

22 Current rabies vaccines IMOVAX® RABIES It was produced by Sanofi Pasteur SA. It is a sterile, stable, freeze-dried suspension

of rabies virus prepared from strain

PM-1503-3M obtained from the

Wistar Institute, Philadelphia, PA. They had harvested the virus from infected human diploid cells, MRC-5 strain, concentrated by ultrafiltration and inactivated by beta-propiolactone. The finished, freeze-dried vaccine was provided for intramuscular administration in a

single dose vial containing no preservative. After reconstitution, immediately 1.0 mL of the vaccine should be administered.

https://dailymed.nlm.nih.gov/dailymed/archives/fdaDrugInfo.cfm?archiveid=82853

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RabAvert® Produced by Novartis vaccines and

diagnostics Obtained by growing the fixed-virus strain

flury LEP (59th passage) in primary cultures of chicken fibroblasts. The virus is inactivated with β-propiolactone, and further processed by zonal

centrifugation in a sucrose density-gradient centrifuge. Rabavert is intended for intramuscular (IM) injection. The vaccine contains no

preservative and should be used immediately after reconstitution with the water for injection (WFI).

http://www.suprememed.com/rabavert-rabies-vaccine-kit-2-5-unit-ml-intramuscular-injection-single-dose-vial-1-ml

24 Currently available rabies vaccines in India

http://www.medlineindia.com/vaccines/rabies_vaccine.htm

Brand Name Composition Company Packing MRP Rs.

ABHAYRAB inj Inactivated rabies vaccine prepared on vero cells HUMAN BIOLOGICAL Vial 254.00

RABIPUR inj2.5 iu inactivated rabies antigen (virus multiplied in chicken fibroblast cell cultures), stabilizer (1dose) TEN-haeacel and 0.1% glutamate q.s.

SANOFI AVENTIS Vial 309.00

RABIVAX inj Anti rabies vaccine on human diploid cells SERUM INT. Vial 322.00

VERORAB inj Each freeze dried vaccine contains 1 dose such that the protective activity is > 2.5 iu RANBAXY Vial 304.00

VEROVAX-R inj Inactivated rabies vaccine prepared on vero cells AVENTIS PASTEUR

0.5ml prefilled syringe

281.00

BERIRAB-P inj Each ml contains human immunoglobulin max. 170mg with rabies antibodies 152 iu, aminoacetic acid as stabilizer 22.5mg

SANOFI AVENTIS 2ml 2225.00

BERIRAB-P inj Each ml contains human immunoglobulin max. 170mg with rabies antibodies 152 iu, aminoacetic acid as stabilizer 22.5mg

SANOFI AVENTIS 5ml 5565.00

FAVIRAB inj Each ml contains 200-400 iu of equine derived anti rabies immunoglobulins RANBAXY 5ml vial 1780.00

IMOGAM inj Human derived anti-rabies immunoglobulin 300 iu per vial RANBAXY 2ml vial 3900.00

IMORAB inj Equine antirabies immunoglobulin AVENTIS PASTEUR 5ml vial 625.00

25 Rabies Vaccines

http://dir.indiamart.com/impcat/rabies-vaccine.html

26 Rabies Immunoglobulins

http://dir.indiamart.com/impcat/rabies-vaccine.html

27 Adverse Reactions to Rabies Vaccines and RIG’sMost common side-effects of rabies vaccines: Systemic reactions such as headache, myalgia, malaise (5-

40%) Mild to moderate local reactions and allergies at injection

site (30-74%)

World Health Organization. (2005). WHO expert consultation on rabies: first report.

28 Challenges

Live attenuated viral vaccines sometimes causes rabies in the inoculated animal by its residual virulence or pathogenic mutation during viral propagation in the body.

Development of attenuated live vaccines needs longer time because the virus vaccine is generally established through serial passages

Inactivated RABV vaccines are generally poor immunogens since they do induce poor potent inflammatory responses needed for effective T-cell and B-cell activation.

Intradermal (ID) injection of vaccine inoculation appears to require less vaccine to be effective than intramuscular (IM), which reduces the cost of treatment. A disadvantage of this method is the increased difficulty in administering successful intradermal injections.

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Parenteral vaccination with tissue culture derived vaccines has been in application since the 1970s, and has been followed extensively in all continents of the world.

They have low levels of side effects, and they can be produced at low cost which have found application in both human and veterinary medicine.

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The major disadvantage for post-exposure prophylaxis is the need for compliance with repeated injections to ensure that treatment is successful.

In practice, this requires multiple trips to vaccination centre which, if not followed, can lead to vaccine failure.

The cost of a full course of vaccination, particularly in parts of Asia and Africa, also remains a problem.

31 Alternate development of Rabies vaccines To date, replication-deficient RABV vectors have been generated in which the

RABV M, P, or G genes have been deleted, The ability to clone the gene coding for the RABV glycoprotein into a plasmid

and then expressing the protein in a range of host systems had led to a number of alternative approaches with the potential for new vaccines against rabies such as RABV glycoprotein expressed on the surface of the vaccinia virus. RABV glycoprotein expressed on the surface of the pox virus. RABV glycoprotein expressed by canine adenovirus.

32 Phosphoprotein gene-deleted rabv vaccines The rabies P serves as a cofactor and regulator protein of the polymerase (L). P gene-deleted vectors did not spread from the periphery to the CNS in rag-2

mice, demonstrating their safety due to their inability to invade neuronal tissue. P gene-deleted RABV induced a tenfold increase in protective efficiency

compared with an inactivated rabies vaccine. In addition, the P gene-deleted vector induced a more balanced IgG antibody

response. But their primary drawback was the relatively slow induction of immunity

making it less appropriate for post-exposure prophylaxis .

33 Modified P gene-deleted RV-based vaccines

Mice immunized with a recombinant RABV expressing two G genes showed an eightfold higher protection compared with protection elicited by a single G gene-expressing vaccine.

The potential exists that the increase gene G expression level by two G genes was the cause of the higher VNA and protection detected

The approach to express two copies of the G gene by a P gene-deleted RABV proved to be beneficial in increasing the speed and magnitude of the anti-RABV G and -RABV N responses

However, gene G expression did not exceed that of the replication-competent RV, even with the additional RV G gene.

Nonetheless, this strategy significantly increased the efficacy of P-deleted vectors for use in both pre- and post-exposure.

34 Matrix protein gene-deleted RV-based vaccines M gene-deleted RABV contains all of the necessary viral machinery

to complete viral gene transcription and genome replication M gene-deleted RABV is able to express antigen over a longer

period of time. M gene-deleted RV was also more immunogenic and provided

superior protection when compared with protection elicited by inactivated vaccines.

35 Generation of highly attenuated rabies recombinant viruses using modified G genes

Pathogenicity lies at the position of 333 position of the G protein which consists of arginine residue.

An exchange of arginine 333 for glutamine / glycine rendered these virus nonpathogenic. Arg- AGA, AGG Glu- GGA, GGG

So research is been carried out replacing the G gene of SPBN with a modified G gene encoding a single amino acid change.

https://www.researchgate.net/figure/11483724_fig1_FIG-1-Construction-of-recombinant-RV-genomes-At-the-top-A-the-SPBN-vector-derived

36 Intracerebral (IC) Administration of Recombinant Rabies Virus Recombinant rabies virus (RABV) expressing granulocyte-macrophage

colony-stimulating factor (GM-CSF) (LBNSE-GM-CSF) resulted in high innate/adaptive immune responses and protection against challenge with virulent rabies.

It was found that intracerebral administration of LBNSE-GM-CSF, protected mice from developing rabies as late as day 5 after infection.

It resulted in significantly higher levels of chemokine/cytokine expression, infiltration of more inflammatory and immune cells into the CNS .

Enhanced blood-brain barrier (BBB) permeability and increase in virus neutralizing antibodies (VNA).

37 Expression of Interferon Gamma by a Recombinant Rabies Virus Using reverse engineering mouse IFN-γ gene was cloned into a

pathogenic rabies virus backbone, SPBN, to produce the recombinant rabies virus designated SPBNγ.

Incorporation of IFN-γ into the rabies virus genome highly attenuated the virus.

In vitro and in vivo mouse experiments show that SPBNγ infection enhanced the production of type I interferons suggesting that IFN-γ production has antiviral effects in rabies.

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Hicks, D. J., Fooks, A. R., & Johnson, N. (2012). Developments in rabies vaccines. Clinical & Experimental Immunology, 169(3), 199-204.

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World Rabies Day is a cooperative global event planned to reduce the suffering from rabies. This day celebrates Dr. Louis Pasteur’s vision of a rabies free world.

40 References Wang, H., Zhang, G., Wen, Y., Yang, S., Xia, X., & Fu, Z. F. (2011). Intracerebral

administration of recombinant rabies virus expressing GM-CSF prevents the development of rabies after infection with street virus. PloS one, 6(9), e25414.

Hicks, D. J., Fooks, A. R., & Johnson, N. (2012). Developments in rabies vaccines. Clinical & Experimental Immunology, 169(3), 199-204.

McGettigan, J. (2010). Experimental rabies vaccines for humans. Expert Rev Vaccines, 9(10),1177-1186.

Dietzschold, B., Schnell, M., & Koprowski, H. (2005). Pathogenesis of rabies. In The World of Rhabdoviruses (pp. 45-56). Springer Berlin Heidelberg.


Sugiyama, M., & Ito, N. (2007). Control of rabies: epidemiology of rabies in Asia and development of new-generation vaccines for rabies. Comparative immunology, microbiology and infectious diseases, 30(5), 273-286.