Isolation and analysis of the molecular epidemiology and zoonotic significance of Mycobacterium tuberculosis in domestic and wildlife ruminants from three states in India.

The majority of tuberculosis cases in ruminants are caused by Mycobacterium bovis (MB). However, in this study, the authors reported the isolation of Mycobacterium tuberculosis (MT) from bovine milk, nasal swabs and post-mortem tissue samples (n = 841) collected from cattle and buffaloes in the states of Telangana, Maharashtra and Gujarat in India in the period from 2010 to 2015. The isolates (n = 7) were confirmed as Mycobacterium due to their growth characteristics and colony morphology in a commercial liquid medium Mycobacterial Growth Indicator Tube (MGIT)™ employing the BD BACTEC™ MGIT™ 960 system and the Löwenstein-Jensen (LJ) medium supplemented with glycerol but not with sodium pyruvate, and BD-DIFCO™ Middlebrook 7H10 agar containing oleic albumin dextrose catalase (OADC). These isolates were initially identified as members of the M. tuberculosis complex (MTC) using a commercial nested polymerase chain reaction (PCR) kit based on the IS6110 MTC specific nucleotide sequence. The isolates were confirmed as MT using three commercial line probe assay kits, were further genotyped, and the spoligotypes identified were of East African Indian (EAI) 3_IND, EAI5, Central-Asian (CAS) 1_DELHI, U and T1 lineages. Two MT isolates from one antelope (Antilope cervipara) andone gazelle (Gazella bennettii) from Gujarat, which were identified previously, were spoligotyped during this study and identified as belonging to EAI3_IND and EAI5 lineages, respectively. The epidemiological significance and zoonotic implications of regional presence and documentation of the same or two differents poligotypes in different species within the family Bovidae as well as humans is discussed.

La majorité des cas de tuberculose chez les ruminants sont dus à Mycobacterium bovis. Néanmoins, les auteurs rapportent les résultats d'une étude réalisée de 2010 à 2015 en Inde (états de Telangana, Maharashtra et Gujarat), au cours de laquelle Mycobacterium tuberculosis a été isolé à partir de lait de vache ainsi que d'écouvillons nasaux et de prélèvements tissulaires postmortem (n = 841) collectés sur des bovins et des buffles. L'appartenance des isolats au genre Mycobacterium a été confirmée par l'observation des caractéristiques de croissance des colonies et de leur morphologie dans un milieu de culture liquide du commerce (Mycobacterial Growth Indicator Tube [MGIT]™ : tube avec indicateur de croissance mycobactérienne) en utilisant l'automate BD BACTEC™MGIT™ 960 et un milieu de Lowenstein-Jensen additionné de glycérol mais sanspyruvate de sodium, ainsi qu'une gélose BD-DIFCO™ Middlebrook enrichie en acide oléique, albumine, dextrose et catalase (OADC). Dans un premier temps, les isolats ont été identifiés comme étant des membres du complexe M. tuberculosisau moyen d'une amplification en chaîne par polymérase nichée ciblant la séquence nucléotidique spécifique IS6110 du complexe M. tuberculosis. Trois kits commerciaux d'analyse de souches ont permis d'identifier les isolats comme étant M. tuberculosis ; il a ensuite été procédé à l'analyse des génotypes des souches de spoligotypes, lesquelles appartenaient aux lignées East African Indian (EAI) 3_IND,EAI5, Central-Asian (CAS) 1_DELHI, U et T1. Les spoligotypes de deux isolats de M. tuberculosis obtenus précédemment, provenant respectivement d'une antilope(Antilope cervipara) et d'une gazelle (Gazella bennettii) de l'état de Gujarat ont été analysés lors de la présente étude et identifiés comme étant respectivement de lignée EAI3_IND et EAI5. Les auteurs analysent l'importance épidémiologique et la portée zoonotique de la présence rapportée dans la région du même spoligotype ou de deux spoligotypes différents chez des espèces différentes de la famille des Bovidés ainsi que chez l'homme.

La mayoría de los casos de tuberculosis que afectan a los rumiantes son causados por Mycobacterium bovis (MB). En este estudio, sin embargo, los autores dan cuenta del aislamiento de Mycobacterium tuberculosis (MT) en muestras de leche, frotis nasales y tejidos obtenidos post-mortem (n = 841) de ganado vacuno y búfalos de los estados de Telangana, Maharashtra y Gujarat (India) entre 2010 y 2015. Se confirmó que los microorganismos aislados(n = 7) eran micobacterias por sus características de crecimiento y la morfología de las colonias cultivadas en medio líquido comercial Mycobacterial Growth Indicator Tube (MGIT)™ empleando el sistema BD BACTEC™ MGIT™ 960 y el medio Löwenstein-Jensen (LJ) suplementado con glicerol, pero no con piruvato sódico, y agar BD-DIFCO™ Middlebrook 7H10 con ácido oleico, albúmina, dextrosa y catalasa (OADC). Mediante una PCR (reacción en cadena de la polimerasa) anidada comercial basada en la secuencia nucleotídica IS6110 específica del complejo, se empezó por determinar que esos microorganismos pertenecían al complejo M. tuberculosis (MTC). Tras confirmar que se trataba de M. tuberculosis empleando tres ensayos comerciales con sondas en línea, se procedió a caracterizar su genotipo, lo que sirvió para identificar espoligotipos correspondientes a los siguientes linajes: East African Indian (EAI) 3_IND, EAI5, Central-Asian (CAS) 1_DELHI, U y T1. Durante el estudio se caracterizaron asimismo los espoligotipos de dos M. tuberculosis aislados previamente a partir de un antílope (Antilope cervipara) y una gacela (Gazella bennettii) de Gujarat, lo que permitió adscribirlos respectivamente a los linajes EAI3_IND y EAI5. Los autores exponen la importancia desde el punto de vista epidemiológico que tiene la presencia comprobada en la región del mismo espoligotipo o de dos espoligotipos diferentes en distintas especies de la familia Bovidae, así como en el ser humano, y las consecuencias que de ahí se siguen por lo que respecta a posibles zoonosis.

Evaluation of immunogenicity and protective efficacy of a liposome containing Brucella abortus S19 outer membrane protein in BALB/c mice.

The use of liposome as an adjuvant and a vaccine carrier has been cited previously in the literature. It has also been shown to be effective in enhancing the immunogenicity of vaccine candidates. BALB/c mice immunized subcutaneously with outer membrane protein (OMP) of Brucella abortus S19 vaccine strain entrapped in a commercial cationic liposome (S19-OMP-liposome) for vaccine delivery, showed enhanced protection (P<0.05) compared to groups of mice inoculated with S19 OMP alone, S19 live B. abortus vaccine and liposome alone, when challenged intra-peritoneally with virulent B. abortus strain 544 at 30 days post-immunization (DPI). The S19-OMP-liposome preparation was found to be safer compared to the live B. abortus S19 vaccine at 15 days post challenge (DPC), as evidenced by the significant difference in spleen weight between S19-OMP-liposome, S19 OMP and S19 live as well as the liposome control groups (P<0.01). Antibody isotype response profiles of the experimental groups indicated that the immune response was Th1 cell mediated. The protective advantage conferred to mice immunized with S19-OMP entrapped in liposome over those immunized with the live B. abortus S19 version, could probably be related to the significantly different response of IgG2b at 30 DPI (P<0.01), IgG2a (P<0.01), IgG2b (P<0.01) and IgG3 (P<0.05) at the DPC stages, respectively.

Direct typing of Canine parvovirus (CPV) from infected dog faeces by rapid mini sequencing technique.

Canine parvovirus (CPV) is a non-enveloped single stranded DNA virus with an icosahedral capsid. Mini-sequencing based CPV typing was developed earlier to detect and differentiate all the CPV types and FPV in a single reaction. This technique was further evaluated in the present study by performing the mini-sequencing directly from fecal samples which avoided tedious virus isolation steps by cell culture system. Fecal swab samples were collected from 84 dogs with enteritis symptoms, suggestive of parvoviral infection from different locations across India. Seventy six of these samples were positive by PCR; the subsequent mini-sequencing reaction typed 74 of them as type 2a virus, and 2 samples as type 2b. Additionally, 25 of the positive samples were typed by cycle sequencing of PCR products. Direct CPV typing from fecal samples using mini-sequencing showed 100% correlation with CPV typing by cycle sequencing. Moreover, CPV typing was achieved by mini-sequencing even with faintly positive PCR amplicons which was not possible by cycle sequencing. Therefore, the mini-sequencing technique is recommended for regular epidemiological follow up of CPV types, since the technique is rapid, highly sensitive and high capacity method for CPV typing.

Evaluation of the Flinders Technology Associates Cards for Storage and Temperature Challenges in Field Conditions for Foot-and-Mouth Disease Virus Surveillance.

Foot-and-mouth disease virus (FMDV) samples transported to the laboratory from far and inaccessible areas for diagnosis and identification of FMDV pose a major problem in a tropical country like India, where wide fluctuation of temperature over a large geographical area is common. Inadequate storage methods lead to spoilage of FMDV samples collected from clinically positive animals in the field. Such samples are declared as non-typeable by the typing laboratories with the consequent loss of valuable epidemiological data. In this study, an attempt was made to evaluate the robustness of Flinders Technology Associates (FTA) cards for storage and transportation of FMDV samples in different climatic conditions which will be useful for FMDV surveillance. Simulation transport studies were conducted using FTA impregnated FMDV samples during post-monsoon (September-October 2010) and summer season (May-June 2012). FMDV genome or serotype could be identified from the FTA cards after the simulation transport studies with varying temperature (22-45°C) and relative humidity (20-100%). The stability of the viral RNA, the absence of infectivity and ease of processing the sample for molecular methods make the FTA cards an useful option for transport of FMDV genome for identification and type determination. The method can be used routinely for FMDV research as it is economical and the cards can be transported easily in envelopes by regular courier/postal systems. The absence of live virus in FTA card can be viewed as an advantage as it restricts the risk of transmission of live virus.

Antigenic and genetic comparison of foot-and-mouth disease virus serotype O Indian vaccine strain, O/IND/R2/75 against currently circulating viruses.

Foot-and-mouth disease (FMD) virus serotype O is the most common cause of FMD outbreaks in India and three of the six lineages that have been described are most frequently detected, namely Ind2001, PanAsia and PanAsia 2. We report the full capsid sequence of 21 serotype O viruses isolated from India between 2002 and 2012. All these viruses belong to the Middle East-South Asia (ME-SA) topotype. The serological cross-reactivity of a bovine post-vaccination serum pool raised against the current Indian vaccine strain, O/IND/R2/75,was tested by virus neutralisation test with the 23 Indian field isolates, revealing a good match between the vaccine and the field isolates. The cross reactivity of the O/IND/R2/75 vaccine with 19 field isolates from other countries (mainly from Asia and Africa) revealed a good match to 79% of the viruses indicating that the vaccine strain is broadly cross-reactive and could be used to control FMD in other countries. Comparison of the capsid sequences of the serologically non-matching isolates with the vaccine strain sequence identified substitutions in neutralising antigenic sites 1 and 2, which could explain the observed serological differences.

Rabies virus isolates of India - simultaneous existence of two distinct evolutionary lineages.

Rabies is a fatal viral disease of serious public health implication. The disease is enzootic in India. In the present study, thirty six rabies virus isolates were obtained from terrestrial mammals of India during 2002-2012. Ecto-domain coding region of the glycoprotein gene from all the isolates were sequenced and the phylogenetic analysis was performed in relation to the global rabies and rabies related virus isolates. The Indian isolates grouped into two distinctly separate lineages with majority of the Indian isolates in Arctic like 1 lineage and the remaining isolates in sub-continental lineage. Isolates of the two distinct lineages were identified simultaneously from the same geographical region. Time scaled phylogenetic tree indicated that the sub-continental lineage of the virus is one of the earliest clade of rabies virus that diverged from bat rabies virus. On the contrary, the Arctic-like 1 lineage of India appeared to be a more recent divergence event. The amino acid sequence comparison revealed that all the major antigenic sites were almost conserved among the Indian isolates whereas few amino acid variations could be identified around site IIa, minor site I and IV. The dN/dS study based on G ecto-domain is in support of the earlier reports of strong purifying selection. In conclusion, it is evident that the Indian rabies virus isolates are of two major distinct lineages with distant phylogenetic and evolutionary relationship.

Transmission of foot-and-mouth disease virus from experimentally infected Indian buffalo (Bubalus bubalis) to in-contact naïve and vaccinated Indian buffalo and cattle.

This study investigated the transmission of foot-and-mouth disease virus (FMDV) from experimentally infected Indian buffalo to in-contact naïve and vaccinated cattle and buffalo. In each of six rooms, two donor buffalo that had been inoculated with FMDV were housed for five days with four recipient animals, comprising one vaccinated buffalo, one vaccinated calf, one unvaccinated buffalo and one unvaccinated calf. Vaccination was carried out with current Indian vaccine strain (O/IND/R2/75) and challenged on 28 days post-vaccination with an antigenically similar strain (O/HAS/34/05). All 12 donor buffalo and the six unvaccinated cattle and six unvaccinated calves developed clinical signs of foot-and-mouth disease (FMD). In contrast, all six vaccinated cattle (100%) and four out of six vaccinated buffalo (66.6%) were protected from disease but all became infected with FMDV. This confirms that buffalo have the potential to spread FMD by direct contact and that vaccination can block this spread. The numbers of animals in the study were too small to determine if the differences in clinical protection afforded by vaccination of cattle and buffalo are significant and warrant a different dose regime.

Development of anti-bovine IgA single chain variable fragment and its application in diagnosis of foot-and-mouth disease.

Recombinant antibody fragments like single chain variable fragments (scFvs) represent an attractive yet powerful alternative to immunoglobulins and hold great potential in the development of clinical diagnostic/therapeutic reagents. Structurally, scFvs are the smallest antibody fragments capable of retaining the antigen-binding capacity of whole antibodies and are composed of an immunoglobulin (Ig) variable light (VL) and variable heavy (VH) chain joined by a flexible polypeptide linker. In the present study, we constructed a scFv against bovine IgA from a hybridoma cell line IL-A71 that secretes a monoclonal antibody against bovine IgA using recombinant DNA technology. The scFv was expressed in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The binding activity and specificity of the scFv was established by its non-reactivity toward other classes of immunoglobulins as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Kinetic measurement of the scFv indicated that the recombinant antibody fragment had an affinity in picomolar range toward purified IgA. Furthermore, the scFv was used to develop a sensitive ELISA for the detection of foot and mouth disease virus (FMDV) carrier animals.

Pre-clinical toxicity & immunobiological evaluation of DNA rabies vaccine & combination rabies vaccine in rhesus monkeys (Macaca mulatta).

BACKGROUND & OBJECTIVES: Pre-clinical toxicology evaluation of biotechnology products is a challenge to the toxicologist. The present investigation is an attempt to evaluate the safety profile of the first indigenously developed recombinant DNA anti-rabies vaccine [DRV (100 µg)] and combination rabies vaccine [CRV (100 µg DRV and 1.25 IU of cell culture-derived inactivated rabies virus vaccine)], which are intended for clinical use by intramuscular route in Rhesus monkeys. METHODS: As per the regulatory requirements, the study was designed for acute (single dose - 14 days), sub-chronic (repeat dose - 28 days) and chronic (intended clinical dose - 120 days) toxicity tests using three dose levels, viz. therapeutic, average (2x therapeutic dose) and highest dose (10 x therapeutic dose) exposure in monkeys. The selection of the model i.e. monkey was based on affinity and rapid higher antibody response during the efficacy studies. An attempt was made to evaluate all parameters which included physical, physiological, clinical, haematological and histopathological profiles of all target organs, as well as Tiers I, II, III immunotoxicity parameters. RESULTS: In acute toxicity there was no mortality in spite of exposing the monkeys to 10XDRV. In sub chronic and chronic toxicity studies there were no abnormalities in physical, physiological, neurological, clinical parameters, after administration of test compound in intended and 10 times of clinical dosage schedule of DRV and CRV under the experimental conditions. Clinical chemistry, haematology, organ weights and histopathology studies were essentially unremarkable except the presence of residual DNA in femtogram level at site of injection in animal which received 10X DRV in chronic toxicity study. No Observational Adverse Effects Level (NOAEL) of DRV is 1000 ug/dose (10 times of therapeutic dose) if administered on 0, 4, 7, 14, 28 th day. INTERPRETATION & CONCLUSIONS: The information generated by this study not only draws attention to the need for national and international regulatory agencies in formulating guidelines for pre-clinical safety evaluation of biotech products but also facilitates the development of biopharmaceuticals as safe potential therapeutic agents.

Genetic and antigenic characterization of Indian foot-and-mouth disease virus serotype O isolates collected during the period 2001 to 2012.

The phylogenetic analysis of VP1 sequences of the 39 type O foot and mouth virus (FMDV) isolates collected from different regions of India during the year of 2001-12 revealed that all isolates belonged to the Middle East - South Asia (ME-SA) topotype. Based on the amount of divergence among the isolates, the viruses were further classified into three distinct lineages namely Ind 2001, PanAsia and PanAsia-2 as well as a minor, unnamed group. Ind 2001 lineage viruses accounted for most of the current type O outbreaks. At the nucleotide level these isolates showed a divergence of 2% to 14% with an average sequence variation of ~9.9%. The serological spectrum of the current vaccine strain was studied by using bovine vaccinate serum (BVS) raised against O/IND/R2/75. All the current field isolates (n=24) were homologous ('r' value 0.4 to 1.0) to the vaccine strain. Examination of the amino acid sequences for selection pressure revealed the positive selection at amino acid sites 13 and 45.

Development and evaluation of a real-time reverse transcription-loop-mediated isothermal amplification assay for rapid serotyping of foot-and-mouth disease virus.

A one-step, real-time reverse transcription-loop-mediated isothermal amplification assay (RT-LAMP) for rapid detection and serotyping of Indian foot-and-mouth disease virus (FMDV) is described. The RT-LAMP assay was found to be 10(3)-10(5) fold more sensitive in comparison with RT-PCR, with a detection limit ranging from 10(-3) to 10(-5) TCID(50) of virus samples of all three serotypes. The RT-LAMP assay and qRT-PCR could detect 100 percent of clinical samples of three serotypes, whereas the RT-PCR detected 69.7% of type O, 58.1% of type A and 60.0% of Asia 1 samples. The qRT-PCR has the same sensitivity as the RT-LAMP. The assay conditions with absence of cross reactivity within the three serotypes of FMDV and FMDV negative samples were established. The RT-LAMP assay could detect 100% of samples stored in FTA(®) cards. In comparison with the performance of the RT-PCR; the RT-LAMP appears to be more sensitive, rapid and specific, with the potential for use as a point-of-care (POC) test, especially in developing countries. The use of FTA(®) cards for the preservation of RNA samples coupled with the RT-LAMP assay for the identification of serotypes may help in achieving improved FMDV serotype identification both in the field and in the laboratory.

Development of foot-and-mouth disease virus (FMDV) serotype O virus-like-particles (VLPs) vaccine and evaluation of its potency.

Foot-and-mouth disease (FMD) is an economically significant viral disease that rampage dairy and other livestock industries in many countries. The disease is being controlled by the use of an inactivated vaccine. However, a recombinant marker vaccine, which avoids the use of live virus, may be an option for the unambiguous differentiation of infected animals from vaccinated animals. A recombinant baculovirus clone containing P1-2A-3C coding sequences of foot-and-mouth disease virus (FMDV) serotype O(1) Manisa was generated. The FMDV structural proteins along with the 3C protease were expressed in Sf9 cells and the generation of virus like particles (VLP) was studied. The recombinant protein was formulated as vaccine using an oil adjuvant, ISA 206 and potency of the vaccine was tested in cattle. The vaccine had a potency value (PD(50)) of 5.01 and most of the vaccinated animals exhibited neutralizing antibody titers after two immunizations.

Plant expressed coccidial antigens as potential vaccine candidates in protecting chicken against coccidiosis.

Coccidiosis is a disease caused by intracellular parasites belonging to the genus Eimeria. In the present study, we transiently expressed two coccidial antigens EtMIC1 and EtMIC2 as poly histidine-tagged fusion proteins in tobacco. We have evaluated the protective efficacy of plant expressed EtMIC1 as monovalent and as well as bi-valent formulation where EtMIC1 and EtMIC2 were used in combination. The protective efficacy of these formulations was evaluated using homologous challenge in chickens. We observed better serum antibody response, weight gain and reduced oocyst shedding in birds immunized with EtMIC1 and EtMIC2 as bivalent formulation compared to monovalent formulation. However, IFN-γ response was not significant in birds immunized with EtMIC1 compared to the birds immunized with EtMIC2. Our results indicate the potential use of these antigens as vaccine candidates.

Typing of canine parvovirus isolates using mini-sequencing based single nucleotide polymorphism analysis.

The antigenic types of canine parvovirus (CPV) are defined based on differences in the amino acids of the major capsid protein VP2. Type specificity is conferred by a limited number of amino acid changes and in particular by few nucleotide substitutions. PCR based methods are not particularly suitable for typing circulating variants which differ in a few specific nucleotide substitutions. Assays for determining SNPs can detect efficiently nucleotide substitutions and can thus be adapted to identify CPV types. In the present study, CPV typing was performed by single nucleotide extension using the mini-sequencing technique. A mini-sequencing signature was established for all the four CPV types (CPV2, 2a, 2b and 2c) and feline panleukopenia virus. The CPV typing using the mini-sequencing reaction was performed for 13 CPV field isolates and the two vaccine strains available in our repository. All the isolates had been typed earlier by full-length sequencing of the VP2 gene. The typing results obtained from mini-sequencing matched completely with that of sequencing. Typing could be achieved with less than 100 copies of standard plasmid DNA constructs or ≤10¹ FAID50 of virus by mini-sequencing technique. The technique was also efficient for detecting multiple types in mixed infections.

Clinical protection, sub-clinical infection and persistence following vaccination with extinction payloads of O1 Manisa Foot-and-Mouth Disease monovalent vaccine and challenge in goats and comparison with sheep.

Small ruminants play an important role in the epidemiology of Foot-and-Mouth Disease (FMD). Small ruminants are vaccinated with one-half or one-third of cattle dose of oil-based or aqueous vaccines respectively. The extinction antigen payload in vaccine for protection in small ruminants is poorly studied. FMD seronegative Nellore sheep (n=30) and Osmanabadi goats (n=30) were vaccinated with different payloads of O(1) Manisa vaccine (0.45-5 µg). Vaccinated and sero-negative unvaccinated sheep (n=6) and goats (n=6) were challenged intradermally into the coronary band with O(1) Manisa virus. The sheep and goats were monitored for signs of FMD and samples were collected for measuring viraemia and virus associated with nasal swabs and probang samples. Clotted blood was collected for serology. Vaccines containing antigen payload up to 0.94 µg protected sheep and goats against challenge. Sheep and goats vaccinated with 0.45 µg antigen payload were poorly protected against challenge. An antigen payload of 0.94 µg was sufficient to offer complete protection and also absence of carrier status. Sheep and goats with no vaccination or with poor sero conversion to vaccination showed sub-clinical infection and became carriers. The results of the study suggest that vaccination offers protection from clinical disease even at a low payload of 0.94 µg and hence one-half of cattle dose of the oil-based vaccine formulations is sufficient to induce protective immune response in sheep and goats. Since no live virus could be isolated after 5 days post challenge from the nasal swab or probang samples even though viral RNA was detected, the risk of these animals transmitting disease was probably very low.

Plant expressed EtMIC2 is an effective immunogen in conferring protection against chicken coccidiosis.

Coccidiosis is an economically important disease affecting poultry industry and remains one of the major problems globally. Developing a cost effective sub-unit vaccine may help mitigate loss in the industry. Here, we report expressing one of the microneme proteins, EtMIC2 from Eimeria tenella in tobacco using Agrobacterium-mediated transient expression. The ability of plant expressed recombinant EtMIC2 in eliciting both humoral and cell-mediated immune responses were measured in the immunized birds. The protective efficacy in the vaccinated birds against a homologous challenge was also evaluated. Birds immunized with plant expressed EtMIC2 showed good sero-conversion, reduced oocyst output and increased weight gain when compared to control birds. Our data indicate that use of plant expressed recombinant EtMIC2 in birds was safe and had the potential in imparting partial protection in chickens against homologous challenge.

Expression and solubilization of insect cell-based rabies virus glycoprotein and assessment of its immunogenicity and protective efficacy in mice.

Rabies is a fatal zoonotic disease of serious public health and economic significance worldwide. The rabies virus glycoprotein (RVG) has been the major target for subunit vaccine development, since it harbors domains responsible for induction of virus-neutralizing antibodies, infectivity, and neurovirulence. The glycoprotein (G) was cloned using the baculovirus expression vector system (BEVS) and expressed in Spodoptera frugiperda (Sf-9) cells. In order to obtain a soluble form of G suitable for experimentation in mice, 18 different combinations of buffers and detergents were evaluated for their ability to solubilize the insect cell membrane-associated G. The combination that involved 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) detergent in lysis buffer 1, formulated with Tris, NaCl, 10% dimethyl sulfoxide (DMSO), and EDTA, gave the highest yield of soluble G, as evidenced by the experimental data. Subsequently, several other parameters, such as the concentration of CHAPS and the duration and temperature of the treatment for the effective solubilization of G, were optimized. The CHAPS detergent, buffered at a concentration of 0.4% to 0.7% (wt/vol) at room temperature (23 to 25°C) for 30 min to 1 h using buffer 1, containing 10% DMSO, resulted in consistently high yields. The G solubilized using CHAPS detergent was found to be immunogenic when tested in mice, as evidenced by high virus-neutralizing antibody titers in sera and 100% protection upon virulent intracerebral challenge with the challenge virus standard (CVS) strain of rabies virus. The results of the mice study indicated that G solubilized with CHAPS detergent retained the immunologically relevant domains in the native conformation, thereby paving the way for producing a cell-free and efficacious subunit vaccine.

Expression of codon optimized major capsid protein (L1) of human papillomavirus type 16 and 18 in Pichia pastoris; purification and characterization of the virus-like particles.

The major capsid protein (L1) of human papillomaviruses (HPV) expressed in heterologous systems assembles into virus-like particles (VLPs). We report cloning and expression of codon optimized HPV L1 genes of the two high-risk HPV types 16 and 18 in methylotropic yeast, Pichia pastoris. The VLPs produced in P. pastoris were subjected to three step purification method involving density gradient centrifugations and size exclusion chromatography. The enriched VLPs were characterized using conformation-specific monoclonal antibodies in ELISA and by transmission electron microscopy. Mice immunized with a bivalent HPV16 and HPV18 VLPs developed high serum antibody titers to both HPV types that persisted for 190 days post vaccination. Serum of mice immunized with the HPV-VLP preparations could neutralize homologous pseudoviruses in an in vitro assays. Our results demonstrate that the L1 proteins expressed in P. pastoris fold properly as evidenced by assembly into VLPs and induction of type-specific neutralizing antibody response in mice. This work constitutes a step towards developing an alternate production platform for generating an affordable HPV vaccine to meet the needs of developing countries.

Zona pellucida-based contraceptive vaccines for human and animal utility.

Contraceptive vaccines can be designed to inhibit (i) production of the gametes (sperm and oocyte), (ii) functions of gametes leading to block in fertilization, and (iii) the gamete outcome (pregnancy). The zona pellucida (ZP) glycoproteins have been proposed as candidates for developing contraceptive vaccines by virtue of their critical role in fertilization. Immunization of non-human primates with either native or recombinant ZP proteins leads to curtailment of fertility, which however is invariably associated with ovarian pathology. To avoid oophoritis, immunogens corresponding to mapped B cell epitopes of ZP proteins that are devoid of 'oophoritogenic' T cell epitopes have been proposed. However, ways to overcome the observed oophoritis associated with the ZP-based contraceptive vaccines are yet to be fully defined. This is essential if their use for control of human fertility is to be considered. Nonetheless, contraceptive vaccines based on ZP proteins have shown very promising results in controlling wildlife population such as wild horses, white-tailed deers, elephants, marsupials, grey seals and dogs, where long term infertility or even permanent sterility is desirable.

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.