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HERP - A SYNTHETIC PEPTIDE INHIBITOR TO HERPES VIRUSES

An inhibitor to DNA viruses human herpes simplex viruses was isolated from the venom of Naja n. kaouthia snake. The purification was carried by fractionating of venom on high pressure liquid chromatography (HPLC) using ion exchange Triza-HCl buffer pH 7.4. The inhibitor having mol. Wt. 13.5 kDa is named Herp, herpes virus inhibitor.

Active domain of the purified homogeneous preparation of natural HERP was identified and isolated. Fragments caused by Trypsin digestion were separated by HPLC. The fragment showing the most inhibitory effect was sequenced for its amino acids. After drug discovery technique a synthetic peptide consisting of ten amino acids was constructed, became synthetic HERP having sequence from the N-terminal: Asn-Leu-Tyr-Gln-Phe-Lys-Asn-Met-Iso-Gln. Synthetic HERP mimics the biological property of inhibition of infectivity of herpes viruses in cell cultures.

Herpes simplex viruses (HSV) type 1 and type 2 are double stranded DNA viruses. The clinical entities attributable to HSV-1 include, (1) Acute herpetic gingivostomatitis, (2) Eczema Kaposi's varicelliform eruption.(3) Keratoconjunctivitis infection of eye (4) Herpes encephalitis.-(5) Herpes labilis-cold sores is most common recurrent disease in the form of oral lesions. HSV-2 is implicated in (1) Genital herpes, of penis or the cervix, vulva and vagina. (2) Neonatal herpes can be transmitted to the newborn during the birth by contact with herpetic lesions in the birth canal. Varicella zoster virus is a double stranded DNA virus and it is morphologically identical with herpes simplex viruses. It is a causative agent for shingles in adults which is characterized by an inflammatory reaction of the posterior nerve roots and ganglia, accompanied by the affected sensory nerves.

Infectivity Inhibition of HSV-1 and HSV-2 Viruses by Synthetic HERP:
For comparison, the natural and the synthetic HERP were tested in Vero cell cultures infected with HSV-1 or HSV-2 viruses. The cells were infected in serial concentrations from 102 to 108, three wells were used for each concentration. After absorption of the virus, the cultures were divided into three groups. Group I received medium containing PBS as a positive control. Group II received medium containing 10 µg/ml of natural HERO, and for group III the medium was incorporated with 10 µg/ml synthetic HERP. The tests were read after six days and TCID/50 were calculated from CPE. The results are seen in table 1.

Table-1. Log Inhibition of infectivity of HSV-1 and HSV-2 viruses in the presence of Natural HERP and Synthetic HERP at the concentration of 10 µg/ml in Vero cells.

The results of table 1 clearly show the inhibition of infectivity of HSV viruses in presence of synthetic HERP was comparable to the natural Herp. The infectivity inhibition for HSV-1 with natural Herp and synthetic HERP was 2.1 and 1.2 respectively. Similarly, the inhibition in the infectivity of HSV-2 by natural HERP was 2.5 versus 1.9 with synthetic Herp. The inhibition of HSV viruses can be higher by increasing the concentration of synthetic HERP.

Currently, there is no effective treatment against the infections caused by herpes viruses. Therefore, the synthetic active HERP has great potential as a therapeutic to treat infections caused by DNA viruses. Currently, tuberculosis (TB) is a major global health problem and HERP is inhibitory to acid fast Tubercle bacilli.

TYPING OF HERPES SIMPLEX VIRUSES

The inhibitor to DNA viruses human herpes simplex viruses type 1 and type 2, was isolated from the venom of Naja n. kaouthia snake. The inhibitor having mol. Wt. 13.5 kDa is named HERP, herpes virus inhibitor. Active domain of the purified homogeneous preparation of natural HERP was identified and isolated. After drug discovery technique a synthetic peptide consisting of ten amino acids was constructed, became synthetic HERP having sequence from the N-terminal: Asn-Leu-Tyr-Gln-Phe-Lys-Asn-Met-Iso-Gln. Syn. HERP mimics the biological property of inhibition of infectivity of herpes viruses in different cell cultures.

Currently there is no effective treatment for infections caused by HHV viruses. Same treatment is used for either types of HHV although it is documented that HHV-2 is more aggressive and pathogenic. As HERP is more inhibitory to HHV-2 CPE , may become a suitable treatment.

The typing of human herpes viruses is routinely done after cultivation of clinical specimen by immunological fluorescent antibody technique, requiring an expensive UV light microscope. It is quite possible to develop a typing test for herpes viruses by using pretreated Vero cells with HERP before infecting with the specimen. If the isolate shows no effect due to HERP in comparison to the control then it is HHV 1 if there is inhibition in CPE then the isolate is HHV 2. The isolation as well as typing of herpes clinical specimen can be accomplished simultaneously. After extensive testing large number of isolates, such a test will be rapid, inexpensive and faster and can be performed in any laboratory with tissue culture set up.

Natural and synthetic HERP selectively CPE inhibitory to HHV-2.

Vero cells pretreated with nat. or syn. HERP overnight showed inhibition of CPE selectively to the standard strain of HHV-2 and no effect on HHV-1. Few clinical isolates for herpes viruses were obtained from Woman's Hospital, Houston. The clinical isolates of herpes virus already identified for the types were tested in Vero cells pretreated with 5 µg/ml HERP overnight. The results are shown in table 1.

Table 1. Effect of HERP on clinical isolates of HHV 1 and
HHV 2 viruses in pretreated Vero cells.

The results showed that th qe CPE of HHV 1 isolates was not affected by overnight pretreatment of Vero cells with 5 µg/ml of HERP. However, the CPE of HHV 2 isolates was inhibited by more than two logs in Vero cells pretreated with HERP. The results strongly suggest that the isolation and typing of HHV strains of clinical specimens can be accomplished simultaneously by using HERP and Vero cells.