Confronting the barriers to develop novel vaccines against brucellosis.

Brucellosis is an important zoonotic disease of nearly worldwide distribution. This pathogen causes abortion in domestic animals and undulant fever, arthritis, endocarditis and meningitis in humans. Currently, there is no vaccine licensed for brucellosis in humans. Furthermore, control of brucellosis in the human population relies on the control of animal disease. Available animal vaccines may cause disease and in some cases have limited efficacy. This article discusses recent studies in the development of recombinant protein, DNA and live-attenuated vaccines against brucellosis. Furthermore, we call the attention of the scientific community, government and industry professionals to the fact that for these novel vaccine initiatives to become licensed products they need to be effective in natural hosts and bypass the regulatory barriers present in several countries.

[Immunogenicity of inactivated subunit adsorbed monovalent vaccine against influenza A/California/7/2009 (H1N1) strain].

The immunogenicity of Pandeflu subunit vaccine against influenza A/California/7/2009 (H1N1) was evaluated in 70 healthy volunteers aged 18 to 60 years. The vaccine was intramuscularly injected twice at an interval of 28 days. Each dose (0.5 ml) contains A(HIN1) influenza virus hemagglutinin (15 +/- 2.2 microg), aluminum hydroxide (Denmark) (0.475 +/- 0.075 microg), and the preservative thiomerosal (merthiolate) (50 +/- 7.5 microg). The level of antibodies was determined in the microneutralization assay. After administration of two doses of the vaccine at a 28-day interval, the geometric mean antibody titer (GMAT) reached 1:21.1 with a further increase to 1:30 (the baseline GMAT) was 1:6.1). The frequencies of seroconversion and seroprotection were 71.4 and 59.2%, respectively; the antibody increase factor was 4.92, which meets the CPMP criteria. The administration of the vaccine did not result in adverse reactions in the postvaccination period.

Yersinia pestis YopE contains a dominant CD8 T cell epitope that confers protection in a mouse model of pneumonic plague.

Septic bacterial pneumonias are a major cause of death worldwide. Several of the highest priority bioterror concerns, including anthrax, tularemia, and plague, are caused by bacteria that acutely infect the lung. Bacterial resistance to multiple antibiotics is increasingly common. Although vaccines may be our best defense against antibiotic-resistant bacteria, there has been little progress in the development of safe and effective vaccines for pulmonary bacterial pathogens. The Gram-negative bacterium Yersinia pestis causes pneumonic plague, an acutely lethal septic pneumonia. Historic pandemics of plague caused millions of deaths, and the plague bacilli's potential for weaponization sustains an ongoing quest for effective countermeasures. Subunit vaccines have failed, to date, to fully protect nonhuman primates. In mice, they induce the production of Abs that act in concert with type 1 cytokines to deliver high-level protection; however, the Y. pestis Ags recognized by cytokine-producing T cells have yet to be defined. In this study, we report that Y. pestis YopE is a dominant Ag recognized by CD8 T cells in C57BL/6 mice. After vaccinating with live attenuated Y. pestis and challenging intranasally with virulent plague, nearly 20% of pulmonary CD8 T cells recognize this single, highly conserved Ag. Moreover, immunizing mice with a single peptide, YopE(69-77), suffices to confer significant protection from lethal pulmonary challenge. These findings suggest YopE could be a valuable addition to subunit plague vaccines and provide a new animal model in which sensitive, pathogen-specific assays can be used to study CD8 T cell-mediated defense against acutely lethal bacterial infections of the lung.

[Construction and expression of a coxsackievirus A16 VP1 gene plasmid which delivered by live attenuated Salmonella].

AIM: To develop a coxsackievirus A16 (Cox A16) VP1 gene plasmid which delivered by live attenuated Salmonella. METHODS: The plasmid which expressed VP1 protein of CoxA16 was constructed by gene recombination. Cellular expression was assessed by Western bloten analysis. Then the recombinant attenuated Salmonella which harboring the plasmid were constructed by electro transformation. RESULTS: CoxA16 VP1 gene sequence was inserted into a eukaryotic expression plasmid. VP1 protein was detected in the culture supernatant. CONCLUSION: The plasmid is constructed successfully and it can be expressed effectively in vitro. The recombinant bacteria are constructed successfully. This has provided a basis for further research of an oral CoxA16 vaccine.

Rationalizing the development of live attenuated virus vaccines.

The design of vaccines against viral disease has evolved considerably over the past 50 years. Live attenuated viruses (LAVs)-those created by passaging a virus in cultured cells-have proven to be an effective means for preventing many viral diseases, including smallpox, polio, measles, mumps and yellow fever. Even so, empirical attenuation is unreliable in some cases and LAVs pose several safety issues. Although inactivated viruses and subunit vaccines alleviate many of these concerns, they have in general been less efficacious than their LAV counterparts. Advances in molecular virology--creating deleterious gene mutations, altering replication fidelity, deoptimizing codons and exerting control by microRNAs or zinc finger nucleases--are providing new ways of controlling viral replication and virulence and renewing interest in LAV vaccines. Whereas these rationally attenuated viruses may lead to a new generation of safer, more widely applicable LAV vaccines, each approach requires further testing before progression to human testing.

Induction of broad cross-subtype-specific HIV-1 immune responses by a novel multivalent HIV-1 peptide vaccine in cynomolgus macaques.

One of the major obstacles in the design of an effective vaccine against HIV-1 is its antigenic variation, which results in viral escape from the immune system. Through a bioinformatics approach, we developed an innovative multivalent HIV-1 vaccine comprised of a pool of 176 lipidated and nonlipidated peptides representing variable regions of Env and Gag proteins. The potency and breadth of the candidate vaccine against a panel of HIV-1 subtypes was evaluated in nonhuman primate (cynomolgus macaques) and humanized mouse (HLA-A2.1) models. The results demonstrate strong immunogenicity with both breadth (humoral and cellular immunity) and depth (immune recognition of widely divergent viral sequences) against heterologous HIV-1 subtypes A-F.

Heterologous prime-boost strategy to immunize very young infants against measles: pre-clinical studies in rhesus macaques.

Infants in developing countries are at high risk of developing severe clinical measles if they become infected during the "window of vulnerability" (age 4-9 months), when declining maternal antibodies do not protect against wild virus, yet impede successful immunization by attenuated measles vaccine. We developed two Sindbis replicon-based DNA vaccines expressing measles virus hemagglutinin and fusion protein with the goal of priming young infants to respond safely and effectively to subsequent boosting with attenuated measles vaccine. Intradermal prime with DNA vaccines by needle-free injection followed by aerosol or parenteral boost with licensed measles vaccine was well tolerated by juvenile and young infant rhesus macaques, and protected against clinical measles and viremia on wild-type virus challenge. A proteosome-measles vaccine administered alone (three doses) or as a boost following DNA vaccine priming was also safe and protective. These promising results pave the way for clinical trials to assess this prime-boost strategy.

Role of RpoS in fine-tuning the synthesis of Vi capsular polysaccharide in Salmonella enterica serotype Typhi.

Regulation of the synthesis of Vi polysaccharide, a major virulence determinant in Salmonella enterica serotype Typhi, is under the control of two regulatory systems, ompR-envZ and rscB-rscC, which respond to changes in osmolarity. Some serotype Typhi strains exhibit overexpression of Vi polysaccharide, which masks clinical detection of lipopolysaccharide O antigen. This variation in Vi polysaccharide and O antigen display (VW variation) has been observed since the initial studies of serotype Typhi. In this study, we report that rpoS plays a role in this increased expression in Vi polysaccharide. We constructed a variety of isogenic serotype Typhi mutants that differed in their expression levels of RpoS and examined the role of the rpoS product in synthesis of Vi polysaccharide under different osmolarity conditions. Vi polysaccharide synthesis was also examined in serotype Typhi mutants in which the native promoter of the rpoS was replaced by an araCP(BAD) cassette, so that the expression of rpoS was arabinose dependent. The RpoS(-) strains showed increased syntheses of Vi polysaccharide, which at low and medium osmolarities masked O antigen detection. In contrast, RpoS(+) strains showed lower syntheses of Vi polysaccharide, and an increased detection of O antigen was observed. During exponential growth, when rpoS is unstable or present at low levels, serotype Typhi RpoS(+) strains overexpress the Vi polysaccharide at levels comparable to those for RpoS(-) strains. Our results show that RpoS is another regulator of Vi polysaccharide synthesis and contributes to VW variation in serotype Typhi, which has implications for the development of recombinant attenuated Salmonella vaccines in humans.

RotaTeq (sanofi pasteur/Wistar Institute/Children's Hospital of Philadelphia).

Sanofi pasteur MSD (a joint venture between Merck & Co and sanofi pasteur (formerly Aventis Pasteur)), in collaboration with the Wistar Institute and the Children's Hospital of Philadelphia, is developing RotaTeq, an oral liquid pentavalent vaccine for the potential prevention of rotavirus infection-related disorders, such as infant diarrhea and malabsorption. Phase III trials had commenced by March 2001.

T cell clones raised from chronically infected healthy humans by stimulation with Toxoplasma gondii excretory-secretory antigens cross-react with live tachyzoites: characterization of the fine antigenic specificity of the clones and implications for vaccine development.

Excreted-secreted Ags (ESA) of Toxoplasma gondii (Tg) play an important role in the stimulation of the host immune system in both acute and chronic infections. To identify the parasite Ag(s) involved in the maintenance of T cell-mediated long term immunity, 40 ESA-specific T cell clones were derived from three chronically infected healthy subjects. All the clones were CD4+ and recognized both ESA and live tachyzoites in a HLA-DR-restricted manner. Conversely, CD4+ tachyzoite-specific T cell clones from the same subjects proliferated in response to ESA, pointing to shared immunodominant Ags between ESA and Tg tachyzoites. By T cell blot analysis using SDS-PAGE-fractionated parasite extracts, the following patterns of reactivity were detected. Of 25 clones, 6 recognized Tg fractions in the 24- to 28-kDa range and proliferated to purified GRA2, 5 reacted with Tg fractions in the 30- to 33-kDa range; and 4 of them proved to be specific for rSAg1. Although surface Ag (SAg1) is not a member of ESA, small amounts of this protein were present in ESA preparation by Western blot. Of 25 clones, 8 responded to Tg fractions in the 50- to 60-kDa range but not to the 55-kDa recombinant rhoptries-2 parasite Ag, and 6 did not react with any Tg fraction but proliferated in response to either ESA or total parasite extracts. In conclusion, CD4+ T cells specific for either ESA (GRA2) or SAg1 may be involved in the maintenance of long term immunity to Tg in healthy chronically infected individuals.

Formulation, stability, and delivery of live attenuated vaccines for human use.

The successful use of live attenuated viral and bacterial vaccines depends not only on the proper choice and delivery of the microorganisms, but also on maintaining the sufficient potency required for an immune response. The inherent lability of live organisms presents a particular formulation challenge in terms of stabilizing and preserving vaccine viability during manufacturing, storage, and administration. This review examines pharmaceutical approaches to the stabilization, formulation, and lyophilization of biological macromolecules in general, as well as the specific applicability of these principles to live attenuated viral and bacterial vaccines. Several formulation development case studies with live vaccines are presented. In addition, comparative stability data are summarized for many other live viral and bacterial preparations. Various pharmaceutical issues with conventional and novel delivery systems for administration of parenteral and oral live vaccines are also discussed.

Influenza vaccines: a main problem in control of pandemics.

The optimal strategy for control of pandemic influenza is early vaccination with influenza vaccine produced from influenza pandemic strains. However, for pandemic control, vaccine improvements are essential and should include quicker ways of manufacturing and testing of vaccine as well as flexibility on the part of licensing bodies. The production of mass doses of monovalent vaccine in a short time can be more realistic if egg independent production technology can be adopted. In this respect production of an influenza vaccine on a stable cell line can solve many of the problems in increased production of influenza vaccine. But the difficulty with influenza vaccines is that the yield of human influenza viruses on tissue culture is much lower than in embryonated eggs. A new high-yield donor is needed for construction of recombinants with a new pandemic strain, which can replicate in a stable cell line with high titre. The live influenza vaccine may be the most appropriate for prophylaxis of influenza pandemic, as the implementation of this vaccine for mass vaccination is simpler than of inactivated influenza vaccine, and this vaccine, after one immunization of unprime persons, induces local mucocosa immunity which plays an important role in the protection against influenza.

Licensing and control authority batch release of influenza vaccine in Germany in accordance with EEC regulations.

The epidemiological situation calls for almost yearly changes in the antigenic composition of influenza vaccine, thus necessitating fresh licensing procedures. Since the time for bringing a new vaccine onto the market should be relatively short, the following work of all parties involved must be done expeditiously: 1) WHO recommendations on new virus strains and their subsequent adaptation by the EEC (February/March); 2) Distribution of the new virus strains to the International Reference Centers for Influenza in the UK and USA (February/March); the centers later issue reference materials for the determination of the haemagglutinin antigen concentration (April/May); 3) Production and testing of seed virus by manufacturers, as well as validation of the producer's inactivation process for the new virus strains (May/June); 4) Licensing of the vaccines by the National Control Authority (Paul-Ehrlich-Institute) (June/July); in the case of previously licensed products, the procedure is limited essentially to the approval of the detailed protocol of production and tests on the new virus strains, clinical studies not being required before licensing because of a lack of time; 5) Paul-Ehrlich-Institute's test for batch release, according to Directive 89/342/EEC, besides protocol approval, conducts material testing of the endotoxin and antigen content of each vaccine lot; the assay for the antigen quantification is especially laborious and sometimes must be repeated because of test invalidity.