Expression of avian influenza virus (H5N1) hemagglutinin and matrix protein 1 in Pichia pastoris and evaluation of their immunogenicity in mice.

The conventional avian influenza vaccines rely on development of neutralizing antibodies against the HA and NA antigens. However, these antigens are highly variable, and hence there is a need for better vaccine candidates which would offer broader protection in animals. The M1 of avian influenza is another major structural protein that has conserved epitopes that are reported to induce CD8+ T cells and can contribute to protection against morbidity and mortality from influenza. Hence in an effort to study the immune response of rM1 either alone or in combination with rHA, the hemagglutinin (HA) and matrix protein (M1) of A/Hatay/2004/H5N1 strain of avian influenza were expressed in Pichia pastoris as his-tagged proteins and purified through Ni-NTA chromatography. The His-tag was removed using TEV protease cleavage site and the immunogenicity of purified rHA and rM1 either alone or in combination was determined in mice. One group of mice was immunized with 5 µg of purified rHA, the other group was immunized with rM1, and a third group of mice were immunized with 5 µg of rHA and rM1. All the animals were boosted twice, once on 28 days postimmunization (dpi) and the second on 42 dpi. The immune response was evaluated by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) assay. The group of mice immunized with rHA and rM1 together showed significantly higher immune response against rHA and rM1 than mice immunized with either HA or M1 antigens. The addition of rM1 with rHA resulted in increased HI titer in animals immunized with both the antigens. These results suggest that the HA and M1 expressed in P. pastoris can be utilized in combination for the development of faster and cost-effective vaccines for circulating and newer strains of avian influenza and would aid in combating the disease in a pandemic situation, in which production time matters greatly.

A validated reverse phase HPLC method for the determination of disodium EDTA in meropenem drug substance with UV-detection using precolumn derivatization technique.

This paper deals with development and validation of a high performance liquid chromatographic method for the quantitative determination of disodium EDTA (Ethylenediaminetetraacetic acid) in Meropenem active pharmaceutical ingredient (API). EDTA was derivatized with Ferric chloride solution by heating at 70 °C in water bath for about 20 minutes and the chromatographic separation achieved by injecting 100 µL of the derivatized mixture into a Waters HPLC system with photodiode array detector using a Phenomenex Luna C18(2) column (250 × 4.6 mm), 5 µ. The mobile phase consisting of 5% methanol and 95% of 0.7 g/L solution of Tetra butyl ammonium bromide and 4.6 g/L solution of sodium acetate trihydrate in water (pH adjusted to 4.0 with the help of acetic acid glacial) and a flow rate of 1 milliliter/minute. EDTA eluted at approximately 6 minutes. The method was suitably validated with respect to specificity, linearity of response, precision, accuracy, ruggedness, stability in analytical solution, limit of quantitation and detection and robustness for its intended use.

Immune Response in Goats to Different Payloads of FMDV Monovalent Vaccine: Protection Against Virulent Challenge and Development of Carrier Status.

The relationship of Foot-and-Mouth Disease virus antigen payload and number of dose of vaccine conferring protection against virus challenge in goats was studied. Goats vaccinated with oil adjuvant Foot-and-Mouth Disease vaccines containing different antigen payloads with or without booster resisted virulent challenge at 21 days post-vaccination or 7 days after booster respectively. However, localized sub-clinical infection was observed in two vaccinated goats on 35 days post-challenge. RNA could be detected from 31.8% of vaccinated goats (10(2.69)-10(4.99) viral RNA copies per cotton swab of nasal secretions) on day 35 post-challenge. Since no live virus could be isolated after 5 days post-challenge, the risk of these animals transmitting the disease was probably very low. The finding showed that oil adjuvant Foot-and-Mouth Disease vaccines containing antigen payload of 1.88 µg may prevent or reduce the local virus replication at the oropharynx and shedding of virus from nasal secretions and thereby reduce the amount of virus released into the environment subsequent to exposure to live virus. This study also showed that goats with poor sero conversion to vaccination can be infected without overt clinical signs and became carriers like sheep.

A Validated Stability-indicating Reverse Phase HPLC Assay Method for the Determination of Memantine Hydrochloride Drug Substance with UV-Detection Using Precolumn Derivatization Technique.

This present paper deals with the development and validation of a stability indicating high performance liquid chromatographic method for the quantitative determination of Memantine hydrochloride. Memantine hydrochloride was derivatized with 0.015 M 9-fluorenylmethyl chloroformate (FMOC) and 0.5 M borate buffer solution by keeping it at room temperature for about 20 minutes and the chromatographic separation achieved by injecting 10 muL of the derivatized mixture into a Waters HPLC system with photodiode array detector using a kromasil C18 column (150 x 4.6 mm), 5 mu. The mobile phase consisting of 80% acetonitrile and 20% phosphate buffer solution and a flow rate of 2 milliliter/minute. The Memantine was eluted at approximately 7.5 minutes. The volume of FMOC used in derivatization, concentration of FMOC and derivatization time was optimized and used. Forced degradation studies were performed on bulk sample of Memantine hydrochloride using acid (5.0 Normal (N) hydrochloric acid), base (1.0 N sodium hydroxide), oxidation (30% hydrogen peroxide), thermal (105 degrees C), photolytic and humidity conditions. The developed LC method was validated with respect to specificity, precision (% RSD about 0.70%), linearity (linearity of range about 70-130 mug/mL), ruggedness (Overall % RSD about 0.35%), stability in analytical solution (Cumulative % RSD about 0.11% after 1450 min.) and robustness.

Experimental transmission of foot-and-mouth disease among Indian buffalo (Bubalus bubalis) and from buffalo to cattle.

Indian buffalo and cattle were infected experimentally with a serotype O strain of foot-and-mouth disease virus of buffalo origin. Whereas intradermolingual inoculation of buffalo produced largely sub-clinical infection, inoculation in the dental pad produced vesicles in the mouth and on the feet. A buffalo infected via the dental pad transmitted infection to cattle and buffalo by direct contact with them for 24h. The contact-exposed buffalo developed (1) delayed-onset clinical signs, and (2) shedding of virus from the nose, commencing before the appearance of vesicles and continuing until the experiment was terminated 10 weeks after exposure. The covert nature of the disease in Indian buffalo, coupled with the prolonged shedding of virus, suggests that this species represents a host of epidemiological importance.