Preserving immunogenicity of lethally irradiated viral and bacterial vaccine epitopes using a radio- protective Mn2+-Peptide complex from Deinococcus.

Although pathogen inactivation by γ-radiation is an attractive approach for whole-organism vaccine development, radiation doses required to ensure sterility also destroy immunogenic protein epitopes needed to mount protective immune responses. We demonstrate the use of a reconstituted manganous peptide complex from the radiation-resistant bacterium Deinococcus radiodurans to protect protein epitopes from radiation-induced damage and uncouple it from genome damage and organism killing. The Mn(2+) complex preserved antigenic structures in aqueous preparations of bacteriophage lambda, Venezuelan equine encephalitis virus, and Staphylococcus aureus during supralethal irradiation (25-40 kGy). An irradiated vaccine elicited both antibody and Th17 responses, and induced B and T cell-dependent protection against methicillin-resistant S. aureus (MRSA) in mice. Structural integrity of viruses and bacteria are shown to be preserved at radiation doses far above those which abolish infectivity. This approach could expedite vaccine production for emerging and established pathogens for which no protective vaccines exist.

Strategies for improved stability of Peste des Petits Ruminants Vaccine.

The main focus of this work was the improvement of the stability of the current PPRV vaccine. First, new formulations based on the Tris buffer were tested, with and without the addition of sucrose and trehalose and compared with the formulation normally used to stabilize the vaccine, the Weybridge medium. The results show a virus half-life of 21 h at 37°C and 1 month at 4°C for the Tris/trehalose liquid formulation and, in the lyophilized form, the formulation was able to maintain the viral titer above the 1 × 10(4) TCID(50)/mL (>10 doses/mL) for at least 21 months at 4°C (0.6 log lost), 144 h at 37°C (0.6 log lost) and 120 h at 45°C (1 log lost). Secondly, a strategy based on culture medium composition manipulation aiming at improving the intrinsic PPRV vaccine stability was also evaluated. The addition of 25 mM fructose resulted in a higher virus production (1log increase) with higher stability (2.6-fold increase compared to glucose 25 mM) at 37°C. Increased concentrations of NaCl, improved virus release, reducing the cell-associated fraction of the virus produced. Moreover this harvesting strategy is scalable and more suitable for a larger scale production than the freeze/thaw cycles normally used. The information gathered in this work showed that it is possible for the PPRV vaccine to have adequate short-term stability at non-freezing temperatures to support manufacturing, short-term shipping and storage. The identification of a more stable formulation should significantly enhance the utility of the vaccine in the control of a PPRV outbreak.

Factors affecting the stability of viral vaccines.

The stability of viral vaccines is determined by the rate of loss of "integrity" of the viral antigen during storage. For live vaccines, such as measles, mumps, rubella, canine distemper, stability is equivalent to the preservation of the infectious titres. For inactivated and subunit vaccines, the preservation of the antigenic structure and the correct steric presentation of the relevant epitopes are the parameters which determine their stability. In general, the following factors may have a negative effect on stability: temperature, pH outside the physiological limits, organic solvents, repeated freezing and thawing, some antiseptics and inactivating agents, and light. However their negative effect is in most cases specific for the individual viruses. Approaches to stabilisation of most vaccines are based on the elimination or neutralisation of the negative factors. Practical examples for the most relevant existing vaccines are described.

[The immunity indices of BALB/c mice immunized with an inactivated antigen of the Machupo virus]. / Pokazateli immuniteta u myshei linii BALB/c, immunizirovannykh inaktivirovannym antigenom virusa Machupo.

The paper presents the data characterizing parameters of specific and nonspecific immunity in BALB/c mice immunized with gamma-ray-inactivated Machupo virus antigen or its formalinized antigen. The gamma-ray inactivated preparation was shown to be more immunogenic for BALB/c mice. A certain relationship between the time course of activity of nonspecific immunity factors in the immunized animals and the protective activity of the preparation under study was also noted. The decisive role of the T-cell part of the immune system was demonstrated in the resistance of this model animal to Machupo virus infection.

The protective antigens of equine herpesvirus type 1.

Equine herpesvirus type 1 was cultivated in swine testis cell cultures and partially purified by differential centrifugation and centrifugation in a linear sucrose density gradient. The viral envelope was separated from the nucleocapsid by treatment with Rexol 25J followed by sucrose gradient centrifugation. The envelope and nucleocapsid preparations were then electrophoresed in polyacrylamide gel after solubilization with sodium dodecyl sulphate. Hamsters were immunized with various preparations of the partially purified virus, including live or inactivated equine herpesvirus type 1 and viral envelope and nucleocapsid, all derived from the Kentucky D strain of the virus. Challenge of the immunized hamsters, with a hamster-adapted strain of equine herpesvirus type 1 demonstrated protection only in those animals which had been vaccinated with envelope-containing materials. When vaccination was carried out with fractions of electrophoresed envelope or nucleocapsid, protection was induced only by polypeptides of high molecular weight containing a glycoprotein component of the envelope of equine herpesvirus type 1.

Immunization of mice against encephalomyocarditis virus. II. Intraperitoneal and respiratory immunization with ultraviolet-inactivated vaccine: effect of Bordetella pertussis extract on the immune response.

Mice were immunized by intraperitoneal (ip) or respiratory administration of ultraviolet-inactivated virus alone or with Bordetella pertussis extract (BPE) as an adjuvant. The effect of immunization was tested by determination of antibody titers and by survival of a lethal challenge with 200 LD(50) of a virulent (large-plaque variant) strain of EMC virus. For plain vaccine the ip 50% effective dose (ED(50)) was 37 hemagglutination units (HAU; ca. 4 x 10(6) plaque-forming unit equivalents); with adjuvant the ip ED(50) was reduced to 20 HAU. After respiratory immunization by intratracheal injection, an ED(50) value of 100 HAU was found, which was not affected by BPE. After ip vaccination the primary immune response was enhanced by BPE, but the challenge response, measured 3 weeks after challenge, was unaffected. Respiratory immunization induced a primary response which was not influenced by BPE, but here the challenge response was enhanced by the adjuvant. After secondary treatment (challenge or booster vaccination) serum antibodies and protection against challenge persisted for at least 1 year.

Immunity to aerosol challenge in guinea pigs immunized with gamma-irradiated Venezuelan equine encephalitis vaccines.

In a previous report, it was shown that nonviable Venezuelan equine encephalitis (VEE) vaccines prepared by exposure of virus suspensions produced in WI-38 cells to ionizing radiations were highly effective in protecting guinea pigs subjected to intraperitoneal (ip) challenge with VEE virus. To characterize further the efficacy of irradiated vaccines, guinea pigs were immunized with three lots of vaccine inactivated by exposure to 8 x 10(6) r of gamma rays and then were challenged via the respiratory route with aerosols of VEE virus. Animals that received a series of three ip inoculations of vaccine at 1-week intervals showed high levels of resistance to aerosol challenge. The 50% effective dose values of vaccines ranged from <0.0016 to 0.0051 ml for respiratory challenge and from <0.00074 to 0.0011 ml for intraperitoneal challenge. Serological studies showed that antigenicity of the irradiated vaccines was excellent. Moderate to high levels of serum-neutralizing and hemagglutination-inhibiting antibodies were demonstrated in the majority of animals vaccinated with undiluted or 10(-1) dilutions of the vaccines. However, serum-neutralizing and hemagglutination-inhibiting antibody levels were not always indicative of the level of immunity, because some animals in which significant antibody could not be demonstrated were able to survive challenge with VEE virus.

Gamma-irradiated Venezuelan equine encephalitis vaccines.

The efficacy of Formalin-inactivated Venezuelan equine encephalitis (VEE) vaccine has been reported to be low for man. Although a live VEE vaccine has been shown to be highly effective for the protection of laboratory workers, local and systemic reactions have occurred in approximately 20% of inoculated individuals. Therefore, studies were initiated in an attempt to produce an inactivated vaccine of high potency with low toxicity. Inactivated VEE vaccines were prepared by exposing virus suspensions to 8 x 10(6) or 10 x 10(6) r of gamma radiation. Irradiated VEE vaccines prepared from virus suspensions produced in Maitland-type chick embryo (MTCE) cell cultures and in monolayer cultures of human diploid strain WI-38 cells were highly immunogenic for mice and guinea pigs. Guinea pigs vaccinated with a series of three inoculations of vaccine (MTCE) survived challenge with at least 10(8.4) mouse intracerebral 50% lethal doses of VEE virus. Irradiated vaccines induced high levels of serum-neutralizing and hemagglutinin-inhibiting antibodies in guinea pigs and rabbits. These findings suggest that ionizing radiation may be effective in the preparation of an inactivated VEE vaccine.