Evaluation of Adenylate Cyclase Toxoid Antigen in Acellular Pertussis Vaccines by Using a Bordetella pertussis Challenge Model in Mice.

Bordetella pertussis is the primary causative agent of pertussis (whooping cough), which is a respiratory infection that leads to a violent cough and can be fatal in infants. There is a need to develop more effective vaccines because of the resurgence of cases of pertussis in the United States since the switch from the whole-cell pertussis vaccines (wP) to the acellular pertussis vaccines (aP; diphtheria-tetanus-acellular-pertussis vaccine/tetanus-diphtheria-pertussis vaccine). Adenylate cyclase toxin (ACT) is a major virulence factor of B. pertussis that is (i) required for establishment of infection, (ii) an effective immunogen, and (iii) a protective antigen. The C-terminal repeats-in-toxin domain (RTX) of ACT is sufficient to induce production of toxin-neutralizing antibodies. In this study, we characterized the effectiveness of vaccines containing the RTX antigen against experimental murine infection with B. pertussis RTX was not protective as a single-antigen vaccine against B. pertussis challenge, and adding RTX to 1/5 human dose of aP did not enhance protection. Since the doses of aP used in murine studies are not proportionate to mouse/human body masses, we titrated the aP from 1/20 to 1/160 of the human dose. Mice receiving 1/80 human aP dose had bacterial burden comparable to those of naive controls. Adding RTX antigen to the 1/80 aP base resulted in enhanced bacterial clearance. Inclusion of RTX induced production of antibodies recognizing RTX, enhanced production of anti-pertussis toxin, decreased secretion of proinflammatory cytokines, such as interleukin-6, and decreased recruitment of total macrophages in the lung. This study shows that adding RTX antigen to an appropriate dose of aP can enhance protection against B. pertussis challenge in mice.

Phase II safety and immunogenicity study of type F botulinum toxoid in adult volunteers.

An aluminum hydroxide (alum)-adsorbed, purified, botulinum F toxoid (Bot F) vaccine was manufactured to be administered as a stand-alone monovalent vaccine or to be added to the current botulinum pentavalent toxoid vaccine to make a hexavalent vaccine. We conducted a phase II trial of the Bot F vaccine over 3 years in 144 healthy adult volunteers to identify one of three vaccination schedules that was safe and maximally immunogenic for adult volunteers. We vaccinated 116 volunteers 1-3 times with Bot vaccine, and 28 volunteers 1-3 times with a licensed, alum-adsorbed hepatitis B vaccine (Engerix-B) as a reaction control group. After 1 year, 42 Bot volunteers with low, mouse anti-toxin titers (<0.10 IU/ml) received a booster injection and were followed for an additional year. The Bot vaccine inoculated three times over 28-42 days was generally well tolerated and safe, whether injected by the subcutaneous (s.c.) or intramuscular (i.m.) route, although it caused significantly more local reactions than did the HBV vaccine. Two vaccination schedules of three primary injections over 42 days (days 0, 14 and 42 or days 0, 21 and 42) provided significantly better protective immunity (anti-toxin levels >0.02 IU/ml) than did vaccinations given over 28 days (days 0, 7 and 28). Vaccine reactogenicity and immunogenicity were similar over 42 days whether administered subcutaneously or intramuscularly. However, even the most immunogenic schedule left 7-16% of volunteers unprotected at day 56 and 33-42% of vaccinees unprotected at 1 year. The booster dose administered at 1 year induced high levels of protective serum anti-toxin in all persons assayed which persisted for at least one additional year. A more potent vaccine formulation will be required to protect more individuals after primary immunization.

Injectable silicone implants as vaccine delivery vehicles.

Injectable silicone implants were assessed as vaccine delivery vehicles in sheep, using either the model antigen avidin or Clostridium tetani and Clostridium novyi toxoids. Two types of implant were compared, the matrix type, that has been shown to deliver antigen in vitro in a first-order profile over approximately 1 month, and the covered rod type, that delivers antigen for several months in a zero-order profile. The implants were prepared using lyophilized antigen and adjuvant (in this case, recombinant ovine interleukin-1beta; rovIL-1beta) and manufactured in the absence of extremes of temperature or pH or the use of organic solvents. Use of the matrix type implant was capable of inducing antibody responses equivalent to those induced by conventional vaccination with aluminium hydroxide adjuvant ("alum"). The use of the covered rod implants, that release very low levels of antigen over a long period, induced responses that were markedly enhanced over the alum control groups. The covered rod implant also favoured production of both IgG1 and IgG2 isotypes in contrast to responses of matrix-vaccinated sheep and conventionally vaccinated control sheep in which IgG1 predominated. Prolonged duration of the antibody response was also observed following vaccination with covered rod implants. Dose-response analysis using the matrix implant demonstrated a trend towards improved responses for lower antigen doses. Clostridial vaccination of sheep showed that protective antibody titres up to 4-fold higher than for alum-adjuvanted groups could be induced by administering the antigen in the covered rod implant. Responses elicited by all implant groups were dependent on the inclusion of adjuvant into the implant formulation.

[The protective properties of the lysosomal form of Pseudomonas aeruginosa anatoxin in a test of its active protection of experimental animals]. / Izuchenie protektivnykh svoistv liposomal'noi formy anatoksina Pseudomonas aeruginosa v teste aktivnoi zashchity éksperimental'nykh zhivotnykh.

The protective activity of the liposomal form of P. aeruginosa toxoid and intact phosphatidylethanolamine liposomes has been evaluated in mice with experimental P. aeruginosa infection. It has been revealed that the liposomal form of P. aeruginosa toxoid, introduced in a single injection, ensures the formation of more intense and prolonged immunity in comparison with that induced by the toxoid adsorbed on aluminum hydroxide. This investigation has revealed that intact phosphatidylethanolamine liposomes also possess protective properties, protecting 10-36.7% of the animals from infection with P. aeruginosa toxigenic strain.

[Immunogenic properties of the liposomal form of Pseudomonas aeruginosa anatoxin]. / Immunogennye svoistva liposomal'noi formy anatoksina sinegnoinoi palochki.

Studies of immunogenic properties of P. aeruginosa anatoxin incorporated into liposomes of varying lipid compositions have shown that single immunization of mice with the anatoxin included into phosphatidyl ethanolamine and lecithin cholesterol liposomes induces a 6-8-fold higher antitoxin antibodies production than immunization with unadsorbed or adsorbed on aluminum hydroxide P. aeruginosa toxoid. The maximal production of antibodies against exotoxin A of P. aeruginosa is observed already on the 14th day. The adjuvant properties of lecithin-cholesterol liposomes were revealed only in the primary immune response, while phosphatidyl ethanolamine-containing liposomes exhibited the capacity for enhancing both the primary and the secondary immune responses. The use of liposomal P. aeruginosa anatoxin made of lipids varying in composition and charge is a promising trend of research aimed at designing new highly effective immunization preparations for P. aeruginosa infection prevention.

The standardization of Cl. perfringens antigens and antisera.

The preparation of laboratory standard antitoxins against Cl. perfringens beta and epsilon toxins is described. These antitoxins are suitable for the quantitative determination of the corresponding antigens by means of the flocculation test. The flocculation test was, however, shown to be more suitable for determining the antigenic value of fresh toxoid rather than toxoid stored without neutralization of excess formalin. A maximal immunity response to alum-precipitated epsilon toxoid was obtained in sheep with two injections containing 90 Lf per dose. The interval between these injections may vary from 2 to 6 weeks. The serum-antibody titres after the primary and secondary injections or after a booster dose given before 12 months after the primary injection did not remain above the protective level in most of the sheep injected for longer than about 5 months. When a sound basic immunity is established the degree of protection following on a booster dose given 12 months later is complete for at least 12 months. An alum-precipitated vaccine containing 25 Lf epsilon toxoid per dose is adequate. The decline in the serum-antibody titre during the first year of vaccination could be eliminated by the use of the antigen in water-in-oil emulsion. Lambs from immune dams were protected for at least up to 13 weeks of age. A satisfactory level of circulating antitoxin against Cl. perfringens beta toxin could be produced in ewes by vaccinating them with APT containing 6.25 Lf beta toxoid per dose. The primary and secondary injections could be separated by 2, 3, 4 or 5 weeks without changing the end result. A booster dose given 2 months before parturition was satisfactory.